Budding Yeast Dbf4 Sequences Required for Cdc7 Kinase Activation and Identification of a Functional Relationship Between the Dbf4 and Rev1 BRCT Domains

DOI: 10.1534/genetics.109.110155

Budding Yeast Dbf4 Sequences Required for Cdc7 Kinase Activation

and Identification of a Functional Relationship Between the Dbf4

and Rev1 BRCT Domains

Victoria Harkins,

1,2

_{Carrie Gabrielse,}

1

_{Louise Haste}

3

_{and Michael Weinreich}

4 Laboratory of Chromosome Replication, Van Andel Research Institute, Grand Rapids, Michigan 49503

Manuscript received September 22, 2009 Accepted for publication October 1, 2009

ABSTRACT

Cdc7-Dbf4 is a two-subunit kinase required for initiating DNA replication. The Dbf4 regulatory subunit is required for Cdc7 kinase activity. Previous studies have shown that the C termini of Dbf4 orthologs encode a single (putative) C2H2zinc (Zn) finger, referred to as ‘‘motif C.’’ By mutational analysis we show that the Zn finger is not required for the essential function of Dbf4. However, deletion and point mutants altering conserved Zn-finger residues exhibit a substantially slowed S-phase, DNA damage sensitivity, and a hypo-mutagenic phenotype following UV irradiation. Using two-hybrid and biochemical assays, we show that the Dbf4 Zn finger interacts with Cdc7 and stimulates its kinase activity. However, a separable Dbf4 region also mediates an interaction with Cdc7 such that only the loss of both Cdc7-interacting regions results in lethality. In contrast, an N-terminal BRCT-like domain is not required for induced mutagenesis nor does it interact with Cdc7. By making chimeric Dbf4 proteins that contain known BRCT domains in

Saccharomyces cerevisiae, we show that the BRCT domain from Rev1, a translesion DNA polymerase, can uniquely substitute for the Dbf4 BRCT domain. Thus, we have mapped regions on budding yeast Dbf4 required for binding and activating Cdc7 kinase. Our data also suggest that the Dbf4 and Rev1 BRCT domains interact with a common protein or structure, although the precise function of both domains and their binding partners remains elusive.

A

two-subunit serine/threonine kinase, Cdc7-Dbf4, is required for initiating DNA replication in all eukaryotes (for reviews, see Sclafani 2000; Bell and Dutta2002). The Dbf4 regulatory protein is required for Cdc7 kinase activation and forms a stable two-subunit complex with Cdc7. Cdc7 protein is expressed at constant levels during the cell cycle, but Dbf4 protein levels are cyclical and peak near the G1/S phase

transition ( Jackson et al. 1993; Cheng et al. 1999; Oshiro et al. 1999; Weinreich and Stillman 1999; Ferreiraet al.2000; Sullivanet al.2008). Since Cdc7 kinase activity parallels Dbf4 protein abundance, it is thought that Dbf4 binding to Cdc7 is the primary mechanism for activating its kinase activity. Numerous data strongly suggest that Cdc7-Dbf4 is recruited to replication origins to promote DNA synthesis by activating the MCM helicase (Hardy and Pautz1996;

Lei et al. 1997; Sato et al. 1997; Weinreich and Stillman 1999; Sheu and Stillman 2006; Francis et al.2009). The MCM helicase is assembled at all origins during G1phase in an inactive form and then is activated

by a Cdc7-Dbf4-dependent step, which triggers origin unwinding and the initiation of DNA synthesis (Geraghty et al. 2000). The precise molecular nature of this step is unknown although Cdc7-Dbf4 kinase is required for origin recruitment of two additional initiation proteins, Cdc45 and the GINS complex, that bind to the MCM helicase (Owenset al.1997; Zouand Stillman2000; Kanemakiet al.2003; Masaiet al.2006; Yabuuchiet al.2006). Cdc45, MCM, and GINS, termed the ‘‘CMG complex,’’ are required for the initiation of DNA synthesis but also travel with the replication fork and are thought to compose the replicative helicase (Gambuset al.2006; Moyeret al.2006; Paceket al.2006). MCM and Cdc45 proteins are in vitro Cdc7-Dbf4 substrates (Weinreich and Stillman 1999; Kihara et al. 2000; Francis et al. 2009), suggesting that phos-phorylation of one or more CMG proteins promotes helicase activation.

The function of the Dbf4 regulatory subunit was discovered following its isolation as a high-copy sup-pressor of thecdc7-1temperature-sensitive (ts) allele in budding yeast (Kitada et al. 1992). Johnston and Thomas(1982) had previously isolateddbf4

temperature-Supporting information is available online athttp://www.genetics.org/ cgi/content/full/genetics.109.110155/DC1.

1_{These authors contributed equally to this work.}

2_{Present address:Zoology Department, Michigan State University, East} Lansing, MI 48823.

3_{Present address: Microbiology Ely, Thermo Fisher Scientific, Ely,} Cambridgeshire CB7 4ET, United Kingdom.

4_{Corresponding author:Van Andel Research Institute, 333 Bostwick Ave.} NE, Grand Rapids, MI 49503. E-mail: [email protected]

sensitive alleles in a screen for new DNA replication mutants. ‘‘Dbf4’’ denotesdumbbellformer4, because, upon elevation to the nonpermissive temperature,dbf4 mutants arrest as large-budded cells or ‘‘dumbbells’’ with incompletely replicated DNA, a common cell-cycle arrest phenotype for DNA replication mutants. Sub-sequently,DBF4orthologs were isolated in Schizosacchar-omyces pombe (dfp11

) (Brown and Kelly 1998), Aspergillus nidulans(nimO) ( Jameset al.1999),Drosophila melanogastor(chiffon) (Landisand Tower1999),Xenopus laevis(XDBF4) (Furukohriet al.2003), mice (MmDbf4) (Lepke et al. 1999), and humans ( Jiang et al. 1999; Kumagaiet al.1999), where it is referred to asHsDBF4or ‘‘ASK’’ foractivator ofS-phase kinase. All Dbf4 proteins bind to the Cdc7 kinase subunit and are required for its kinase activity. Multiple sequence alignment of Dbf4 proteins across species revealed low sequence conserva-tion except in three regions, referred to as motifs N, M, or C, denoting their position within the protein (Figure 1) (Masaiand Arai2000).

Genetic and comparative sequence-based evidence suggests that Dbf4 orthologs encode a single BRCT-like domain that overlaps motif N (Masaiand Arai 2000; Gabrielseet al. 2006). BRCT domains are present in many proteins that respond to DNA damage and were first discovered as a tandemly repeated sequence in the C terminus of the breast cancer susceptibility gene BRCA1 (Koonin et al. 1996), although they are also present in single copy (Borket al.1997). Tandem BRCT domains have been shown to bind phosphorylated proteins (reviewed by Glover et al. 2004). In the absence of structural information on any Cdc7-Dbf4 protein, it is not known if this Dbf4 region resembles a BRCT fold. Since deletion of this region in budding and fission yeasts results in sensitivity to a wide variety of DNA-damaging agents, it is thought that this region of Dbf4 plays a role in the response to DNA damage or replication fork arrest (Ogino et al.2001; Funget al. 2002; Gabrielse et al. 2006). Indeed, Dfp1 has been shown to interact with Swi1 (budding yeast Tof1), which is a component of the replication fork, suggesting that Dbf4 might be targeted to replication forks (Matsumotoet al. 2005). InSaccharomyces cerevisiae, however, mutants that precisely delete only the BRCT-like domain or more extensive N-terminal deletion mutants that restore a deleted nuclear localization sequence (NLS) exhibit little DNA damage sensitivity and are only moderately sensitive to replication fork stalling by the drug hydroxyurea (HU) (Gabrielseet al.2006). Interestingly, loss of the BRCT-like sequence is synthetically lethal withrad53-1 (encod-ing the Chk2 checkpoint kinase) and confers a defect in activating late vs. early replication origins (Gabrielse et al.2006). Together, these and other findings suggest that the BRCT-like domain does not play a prominent role in DNA damage repair under normal circumstances but may instead regulate kinase targeting to early vs. late origins. In addition, Dbf4 may have a redundant role at

stalled replication forks or may participate in a Rad53-mediated checkpoint under conditions yet to be defined. Interestingly, nearly 30 years agocdc7 mutants were reported to be defective in induced mutagenesis (also known as error-prone repair, one pathway of post-replicative repair, or PRR) (Njagiand Kilbey1982a,b). It is now known thatCDC7functions in theRAD6epistasis pathway for PRR (Pessoa-Brandaoand Sclafani2004). Error-prone repair occurs when the replicative DNA polymerase encounters a bulky lesion that cannot be accommodated within the polymerase active site, the lesion is by-passed and instead replicated across by lower-fidelity translesion (TLS) DNA polymerases such as Rev1 or Rev3-7 (Polz) (reviewed by Waterset al.2009). It is not known how Cdc7-Dbf4 promotes error-prone repair or whether Dbf4 targets the kinase to sites of replication fork stalling or directly to DNA lesions.

Dbf4 binds to Cdc7 to activate its kinase activity. A detailed mutational analysis ofS. pombe Dfp1 revealed that Dfp1 binds to the Hsk1 (Cdc7) subunit using motifs M and C (Oginoet al.2001). This analysis also revealed that there is a spacer region between motifs M and C (150 residues) that is not required for the mitotic function of Dfp1. Hsk1 kinase activity was stimulated by wild-type Dfp1 but was compromised by using Dfp1 mutants lacking motif M or motif C (Oginoet al.2001; Funget al.2002), and mutations within motif M caused methyl methanesulfonate (MMS) and HU sensitivity (Takeda et al. 1999). Evidence from two-hybrid assays also indicated thatS. cerevisiaeDbf4 could interact with Cdc7 using residues spanning motifs M or C (Dowell et al.1994; Hardyand Pautz1996). Human ASK also uses motifs M and C to interact with Cdc7 (Satoet al. 2003). The short, 40-residue region known as motif M does not encode a known protein domain; however, motif C encodes a C2H2-type Zn finger (Masaiand Arai

2000). Although initially defined as DNA-binding mod-ules, there are.20 types of Zn fingers that are involved in protein–protein interactions, DNA binding, RNA binding, ubiquitin binding, and other functions (re-viewed by Brayerand Segal2008).

domains in budding yeast, we find that the Rev1 BRCT domain functionally substitutes for the Dbf4 BRCT-like domain. Paradoxically, although deletion of the Dbf4 N-terminal BRCT-like domain does not result in an error-prone repair defect, we find that the BRCT domain from a known translesion DNA polymerase suppresses the associated defects in Dbf4 activity. Since the BRCT domain from mouse Rev1 promotes targeting to repli-cation foci (Guoet al.2006), this raises the possibility that yeast Rev1 and Dbf4 are targeted to the replication fork via their respective BRCT domains and may interact with the same protein.

MATERIALS AND METHODS

Construction of yeast strains, plasmids, baculoviruses, and growth media:All yeast strains (with the exception of PJ69-4A and KKY02C) are derivatives of W303 (R. Rothstein, Columbia

University) or were backcrossed at least four times to W303 from the parental strain and are listed in Table 1. Genetic manipulation and transformation of yeast was done using standard techniques. YPD denotes rich medium containing 1% yeast extract, 2% peptone, and 2% glucose. We used FOA, a synthetic complete medium containing 1 mg/ml 5-fluoroorotic acid. Drugs were added directly to media before pouring at the indicated concentrations.

All plasmids used in this study are listed in the supporting information, Table S1. DBF4deletions and point mutations were constructed by QuikChange (Stratagene, La Jolla, CA) on pMW489. pMW489 is pRS415 containing DBF4 on a genomic 2.5-kbMluI–XbaI fragment. For each mutation, the entireDBF4sequence on the plasmid was verified by sequenc-ing. BRCT-Dbf4 chimeras were constructed by amplifying single or tandem BRCT repeats from genomic DNA (Bork et al.1997) and were cloned into pCG29 (dbf4-ND221) on an

NcoI fragment. pVMH14 (CHS5) contains residues 161–273, pCG46 (DNL4, 682–942), pCG48 (DPB11, 1–228), pCG165 (DPB11, 306–592), pCG95 (FCP1, 495–595), pCG94 (NOP7, 350–454), pCG51 (POL4, 1–125), pCG45 (RAD9, 1027–1297), TABLE 1

Yeast strains

Strain Relevant genotype Source

W303-1A MATaade2-1 ura3-1 his3-11,-15 trp1-1 leu2-3,-112 can1-100 R. Rothstein PJ69-4A MATatrp1-901 leu2-3,112 ura3-52 his-200 gal4D

gal80DLYS2TGAL1-HIS3 GAL2-ADE2 met2TGAL7-lacZ

E. Craig

KKY02C MATaleu2-3,112 trp1-289 ura3-52 CDC5TURA3 A. Sugino

M895 W303MATadbf4DTkanMX6[pMW490,DBF4 URA3] Gabrielseet al.(2006)

M1016 M895 [pMW489,DBF4 LEU2] Gabrielse_{et al.}(2006)

M1173 M895 [pCG29,dbf4-ND221 LEU2] Gabrielseet al.(2006)

M1494 M895 [pCG54,dbf4-ND2213REV1 BRCT LEU2] This study

M1495 M895 [pCG56,dbf4-ND2213RAP1 BRCT LEU2] This study

M1521 M895 [pCG51,dbf4-ND2213POL4 BRCT LEU2] This study

M1845 M895 [pCM18,dbf4-ND2213rev1-K216M LEU2] This study

M1891 M895 [pCG94,dbf4-ND2213NOP7 BRCT LEU2] This study

M1892 M895 [pCG96,dbf4-ND2213RFC1 BRCT LEU2] This study

M1917 MATaade2-1 ura3-1 his3-11,15 leu2-3,112 can1-100 trp1-289 This study

M1921 M895 [pVMH3,dbf4-CC661,664AA LEU2] This study

M1923 M895 [pVMH5,dbf4-HH674,680LL LEU2] This study

M1937 W303MATadbf4DTkanMX6 TRP1[pMW490, DBF4 URA3] This study

M1978 M895 [pVMH16,dbf4-CD418 LEU2] This study

M1979 M895 [pVMH18,dbf4-CD511 LEU2] This study

M1980 M895 [pVMH21,dbf4-CD575 LEU2] This study

M1981 M895 [pVMH22,dbf4-CD598 LEU2] This study

M1982 M895 [pVMH23,dbf4-CD629 LEU2] This study

M1983 M895 [pVMH7,dbf4-CD654 LEU2] This study

M2025 W303MATadbf4DTkanMX6 trp1-289[pMW490, DBF4 URA3] This study

M2040 M2025 [pMW489, DBF4 LEU2] This study

M2098 M895 [pGR5,dbf4-ND2213rev1-W231L LEU2] This study

M2183 M2025 [pVMH23,dbf4-CD629 LEU2] This study

M2186 M2025 [pVMH3,dbf4-CC661,664AA LEU2] This study

M2394 M895 [pLVH56,dbf4-C664A LEU2] This study

M2395 M895 [pLVH55,dbf4-S677A LEU2] This study

M2396 M895 [pLVH58,dbf4-CD689 LEU2] This study

M2400 W303MATadbf4-6(HH674,680LL) This study

M2402 M895 [pLVH59,dbf4-S677E LEU2] This study

M2403 M895 [pLVH60,dbf4-HH674,680AA LEU2] This study

M2484 M895 [pCG122,dbf4-ND221 x rev1-G193A LEU2] This study

M3361 M2025 [pVMH97,dbf4-P277L LEU2] This study

pCG56 (RAP1, 121–209), pCG54 (REV1, 160–251), and pCG96 (RFC1, 148–243).

Baculovirus transfer plasmids encoding DBF4 deletions (on anNcoI–NotI fragment) were constructed in pAcSG2, and baculoviruses were generated using the Baculo Gold kit (BD Biosciences), plaque purified, and then amplified to high titer. The wild-type DBF4 andHA-CDC7 viruses have been described previously (Weinreichand Stillman1999). The dbf4-6 mutant (HH674,680LL) was integrated at the

dbf4DTkanMX6locus of M895 [dbf4D/pMW490 (DBF4 URA3)] by cotransformation of aHindIII–XbaI fragment containing full-lengthdbf4-HH674,680LL together with pRS415. Leu1

trans-formants were replica plated to FOA. Multiple FOA-resistant colonies were recovered to YPD plates and then tested on YPD plates containing 0.2 mg/ml geneticin to score loss of the

dbf4DTkanMX6 marker. The resulting GenS candidates were confirmed as correct recombinants following PCR amplification of theDBF4locus, tested for temperature sensitivity, and back-crossed to W303.

Cell-cycle analysis and preparation of whole-cell extracts: FACS analysis of DNA content was as described (Weinreich and Stillman1999). Whole-cell extracts were prepared using a trichloroacetic acid extraction method from 10 ml of cells (Foianiet al.1994). Five percent of the whole-cell extract was separated on a 10% or 15% SDS–PAGE gel, blotted, and probed with rabbit polyclonal antisera against GST-Cdc7 (1:4000), GST-Dbf4 (1:1000) (Weinreich and Stillman 1999), or yeast anti-Gal4AD (1:1000, Upstate Cell Signaling) followed by anti-rabbit HRP (1:10,000) in 13 phosphate-buffered saline containing 0.1% Tween and 1% dry milk. To detect Dbf4 proteins in Figure 5A (lanes 1–7), we used a rabbit polyclonal antisera raised against an N-terminal Dbf4 peptide spanning residues 142–153. Ponceau S staining of the blot con-firmed equal protein loading in the samples being compared. UV reversion assay: The procedure was derived from Lawrence and Christensen (1976) and other sources. Briefly, triplicate cultures containingtrp1-289were grown in YPD media to saturation and adjusted to the same number of cells per milliliter in water. Cells were diluted, irradiated with increasing UV fluences up to 80 J/m2_{, and plated onto SCM} complete plates to score survival or onto SCM Trp plates to score TRP1colonies. Plates were kept in the dark for 3 days at 30°(4 days at 25°for thedbf4-1ts mutant) and then counted. Reversion frequencies were calculated relative to surviving cells at each UV fluence, averaging two independent experi-ments (i.e., six measurements).

Immunoprecipitation and kinase assay of Cdc7-Dbf4 pro-tein from Sf9 cells:Sf9 cells were co-infected at an MOI of 10 with baculoviruses expressing HA-Cdc7 and Dbf4 derivatives. After 48 hr, soluble whole-cell extracts were prepared and immunoprecipitated using a 12CA5 monoclonal antibody against the HA epitope. After extensive washing, the immu-noprecipitation (IP) was split in half. Half of the IP was incubated for 30 min at 30°in kinase buffer (50 mmTris–HCl, pH 7.5, 10 mm MgCl₂, 1 mm DTT, 0.1 mm ATP, 10 mCi [g-32_{P]ATP). These samples were then separated on parallel} 10% SDS–PAGE gels and either blotted and probed with Cdc7 and Dbf4 polyclonal antisera, as described above, or subjected to autoradiography.

RESULTS

Motif C is not essential for Dbf4 activity but is required for growth under stress conditions: To de-termine the Dbf4 C-terminal residues that were essential for activity, we tested a series of truncation and point

mutants spanning the C-terminal 392 amino acids of Dbf4 for their ability to rescue the viability of adbf4DTkanMX6 yeast strain by plasmid shuffling (Figure 1). Interestingly, all of the deletion mutants up to residue 418 behaved similarly in that they complemented viability but grew more poorly at 25° than wild type and were strictly temperature sensitive at 37°(Figure 1, B and C). Dele-tions that removed C-terminal residues up to 357 or 312 did not complement viability, indicating that they re-moved essential Dbf4 residues (Figure 1B). An internal deletion removing residues 312–418 also did not com-plement viability (not shown), indicating that these residues were indeed essential for Dbf4 activity and not just when the remainder of the C terminus (including motif C) was also deleted. In addition, all of the viable C-terminal deletion mutants were substantially more sensitive to the DNA-damaging agents HU, bleomycin, and MMS that cause replication fork arrest, double-strand breaks, and alkylation damage, respectively (Figure 1C).

The Zn-finger domain becomes essential when combined with additional perturbations in Dbf4 structure: To determine if the conserved ‘‘motif N’’ and motif M regions were required for Dbf4 activity in the absence of a functional Zn-finger domain, we combined several C-terminal truncations as well as a CC661,664AA mutant affecting the conserved cysteines in the Zn finger with mutations affecting motifs N or M. We found that thedbf4-1point mutation P277L within motif M and both N-terminal and internal deletions removing the BRCT-like region were lethal in combina-tion with all C-terminal mutacombina-tions (Figure 2). Unex-pectedly, although an N-terminal deletion removing the first 65 amino acids of Dbf4 was tolerated, a further deletion to amino acid 109 was also lethal in combina-tion with the C-terminal mutants. The dbf4-ND109 mutant retains the BRCT-like sequence (including motif N) and motif M, has essentially wild-type growth characteristics, kinase activity, and S-phase progression and activates replication origins as does the wild type (Gabrielseet al.2006). As far as we have determined, this mutant behaves exactly as does the wild type with the exception that it removes a nonessential region that interacts with Polo kinase to inhibit mitotic exit (Miller et al. 2009). Therefore, it is possible that, because of their slowed S-phase (see below), the C-terminal mu-tants activate fewer replication origins and depend upon G2/M checkpoint mechanisms to delay entry into

or exit from mitosis. In any event, further moderate perturbations of Dbf4 function cause lethality in the absence of the Zn-finger domain, probably due to a significant defect in kinase activation (see below).

The Dbf4 Zn finger most closely resembles a classical C2H2-type domain and physically interacts

are absolutely conserved as are several additional residues such as G659, E662, F683, and D694 (Figure 3A, blue type), as shown previously (Masai and Arai 2000). A number of additional residues are also highly conserved as well as an S/T residue between the two histidines. DNA-binding Zn-finger proteins typically contain several tandem fingers with the consensus (F/ Y)-X-C-X(2-5)-C-X3-(F/Y)-X5-C-X2-H-X(3-5)-H, whereCis

a hydrophobic residue (Wolfe et al. 2000). However, Dbf4 contains a single Zn finger with the consensus G-Y-C-E-X-C-X3-(F/Y)-X2-C-E-X-H-X5-H-X2-F. This

consen-sus resembles the C2H2-type DNA-binding Zn finger

with several important differences: (1) Dbf4 orthologs have a 9-amino-acid spacing between the last C and first H (instead of 12); (2) they have an aromatic residue immediately preceding the first C (instead of two residues before); and (3) Zn fingers that bind DNA typically have conserved basic and acidic residues that bind the phosphate backbone and bases, but these are not in the correct positions for the Dbf4-type Zn finger (Wolfeet al.2000), suggesting that it might not mediate an interaction with DNA. On the basis of crystallo-graphic information there are at least eight different structural classes of Zn fingers that function as protein– protein interaction domains and at least three of these bind the small-protein modifier ubiquitin (Krishna et al.2003). Inspection of these classes reveals that the Dbf4 Zn finger most closely resembles a classical TFIIIA-type Zn-finger domain; however, on the basis of the reasons cited above it might bind a protein instead of DNA.

Since Dbf4 contains a C2H2-type Zn finger that

defines motif C, we tested the importance of conserved residues within the Zn finger for Dbf4 activity. We made two point mutants that altered either both cysteine or both histidine residues within the Zn finger and tested their ability to complement the strain deleted forDBF4. The point mutants complemented the dbf4D but had phenotypes that were essentially as severe as the C-terminal deletion mutants (Figure 3B). The CC661, 664AA and HH674,680LL mutants were temperature sensitive and extremely sensitive to bleomycin and MMS, although they showed only a mild HU-sensitive pheno-type. An HH674,680AA mutant and a single C664A mutant had the same phenotypes, indicating that the conserved Zn-binding residues are critical for Dbf4 activity. A small 15-residue C-terminal deletion (D689-704) beyond the C2H2 cluster gave the same pattern of sensitivities,

indicating that the entire conserved region of 40 residues is important for Dbf4 activity. Finally, it was possible that the highly conserved S/T residue (S667 in budding yeast) between the two histidine residues could function as a phosphorylation site to impact Dbf4 activity. However, mutation of S677 to Ala or Glu (a phospho-mimic) had no effect on Dbf4 growth or sensitivity to DNA-damaging agents (Figure 3B).

We have described new dbf4 temperature-sensitive alleles, so it is informative to compare these to the previously describeddbf4ts alleles:dbf4-1,dbf4-2,dbf4-3, dbf4-4, anddbf4-5(Kiharaet al.2000). Thedbf4-1,dbf4-2, anddbf4-3alleles have mutations within motif M: P277L, P308L, and P308S, respectively. However, dbf4-4 con-Figure1.—The Dbf4 Zn finger is required for growth under stress condi-tions. (A) Schematic of Dbf4 showing the BRCT-like domain and motifs N, M, and C together with a de-scription of the C-terminal mutants. (B) Single-copy plasmids (LEU21₎

contain-ing the indicated Dbf4 C -terminal deletions were transformed into M895 [dbf4D/pMW490 (DBF4 URA3)] and tested for their ability to complementdbf4D

tains a truncating mutation at residue 596 anddbf4-5 contains a E415K mutation in addition to a W112 nonsense mutation (TGG. TAA) that is predicted to allow formation of an N-terminally truncated protein beginning at the M120 codon but containing E415K. In summary, our newdbf4ts alleles result from the loss of a functional Zn finger either by point mutation or by a deletion analogous to that found in dbf4-4. Further-more, residues 312–418 are essential for Dbf4 function, suggesting that the E415K mutation within thedbf4-5ts allele affects that (unknown) function.

We next determined whether the Zn-finger domain mutants behaved similarly on the chromosome. Since thedbf4-4mutant encodes a representative C-terminal truncation and gives a ts phenotype, we integrated the dbf4-HH674,680LL point mutant at the endogenous DBF4locus. This allele (that we now refer to asdbf4-6) gave very similar phenotypes to the allele when present on a plasmid (Figure 3C), including a tight ts pheno-type. The temperature and DNA damage sensitivities of dbf4-6 were complemented by wild-type DBF4 as ex-pected but also by high-copy CDC7 (Figure 3C). This raises the possibility that mutations in the Dbf4 Zn finger affected the interaction with Cdc7 because in-creasedCDC7copy number rescued thedbf4-6 pheno-types. Below we show that the Zn finger interacts with Cdc7.

Dbf4 C-terminal mutations cause a significant delay in S-phase progression and are defective in UV-induced mutagenesis: We tested whether mutations within the Dbf4 C terminus affected S-phase progression by flow cytometric analysis of DNA content. Dbf4 is required for the initiation of DNA replication, and mutations

affect-ing the essential function of eitherCDC7orDBF4affect entry into and progression through S-phase ( Johnston and Thomas1982; Woodand Hartwell1982). Cdc7-Dbf4 also affects S-phase progression since it is needed throughout S-phase to activate DNA synthesis from individual replication origins (Bousset and Diffley 1998; Donaldson et al.1998). We arrested cells in G₁ phase by using mating pheromone and released cells into fresh medium at the permissive temperature of 25°. Wild-type cells initiated DNA replication between 20 and 30 min following the release and had completed S-phase by 50 min (Figure 4B). Thedbf4-CC661,664AA anddbf4-CD629mutants delayed entry into S-phase to between 30 and 50 min following the G1release and did

not complete S-phase until 60 or 75 min following the release, respectively. These data indicate that the Dbf4 Zn finger is required for normal DNA replication, almost certainly by affecting the ability of Cdc7-Dbf4 to initiate DNA replication.

In contrast to the MMS and bleomycin sensitivity,dbf4 mutants affecting the Zn finger were not particularly sensitive to UV irradiation up to 80 J/m2 _{(Figure 4A,} inset). Deletions that removed the N-terminal 65 or 221 residues of Dbf4 also did not result in UV sensitivity (not shown). We then asked whether the BRCT-like or Zn-finger domains might be required for UV-induced mutagenesis since cdc7 hypomorphic mutants are de-fective in this process (Njagiand Kilbey1982a,b). For this purpose, we measured reversion of the ochre trp1-289 allele to Trp11 prototrophy over a range of UV doses. UV-induced cyclobutane prymidine dimers and 6-4 photoproducts are repaired by the DNA transle-sion polymerases Rev1 and Polhin an error-prone and Figure _{2.—The Dbf4 C} terminus is essential if Dbf4 activity is further impaired. A CC661,664AA point mutation or C-terminal deletions were combined with additional mutations in

DBF4 as indicated, and the resulting mutants were tested for their ability to comple-ment the viability defect of

error-free manner, respectively (reviewed by Prakash et al.2005). Error-prone repair can result in substitution of a sense codon for the nonsense codon oftrp1-289, which in this system results in an2000-fold increase in Trp11 reversion events at 80 J/m2 _{(Figure 4A).} In-terestingly, UV-induced Trp11reversion was abolished by deletion of the Dbf4 C terminus and was substantially diminished by the CC661,664AA mutant. In contrast, an internal deletion (D118-221) that removed the BRCT-like domain or thedbf4-1(P227L) allele within motif M had Trp11 reversion frequencies similar to the wild type. These data indicate that the Dbf4 Zn finger is required for UV-induced mutagenesis but the BRCT-like domain is dispensable for this activity.

Two separable Dbf4 regions direct binding to and activation of the Cdc7 kinase:To determine the regions of Dbf4 required for binding Cdc7 and activating its kinase activity, we expressed HA-Cdc7 together with wild-type or mutant Dbf4 proteins in Sf9 cells. We immunoprecipitated the Cdc7 subunit using an antibody against the HA tag and then examined co-IP of the Dbf4 subunit and also measured Cdc7-Dbf4 kinase activity in the IP (Figure 5A). We previously showed that N-terminal Dbf4 deletions through residue 292 (that partially deletes motif M) bound Cdc7 efficiently and showed little change in Cdc7-Dbf4 auto-phosphorylation or Mcm2 phosphor-ylation (Gabrielse et al. 2006). Cdc7 kinase activity is measured on HA-Cdc7-Dbf4 bound to beads, and we have

noted that Mcm2 phosphorylation (but not Cdc7-Dbf4 auto-phosphorylation) is diminished compared to that seen with the free kinase. In our experimental system, there was no Cdc7 kinase activity in the absence of Dbf4 expression (compare lanes 2 and 3 in Figure 5A). The C-terminal deletions to residue 629 or 418 did not affect Dbf4 binding to Cdc7 but dramatically lowered its kinase activity (lanes 4 and 5). This suggests that the Zn-finger domain plays a substantial role in Cdc7 kinase activation but is not absolutely required for Dbf4-Cdc7 binding. A further deletion mutant to residue 312 (that retains an intact motif M) eliminated the ability of Dbf4 to bind Cdc7 and stimulate its kinase activity (lane 6). There is a critical region between residues 312 and 418 that pro-moted Dbf4 binding to Cdc7 since Dbf4 containing a 312–418 internal deletion also bound very poorly to Cdc7 compared to the wild type and stimulated very little Cdc7 kinase activity (lane 7). The CC661,664AA mutation in the Dbf4 Zn-finger domain did not affect the ability of Dbf4 to bind Cdc7 in the context of full-length Dbf4 but strongly decreased Cdc7-Dbf4 kinase activation (lane 8). The CC661,664AA mutation, however, did affect the ability of Dbf4 to bind Cdc7 when the N-terminal 292 amino acids were deleted, including most of motif M (lane 10). Finally, deletion of the N-terminal 357 amino acids abolished the ability of Dbf4 to bind Cdc7 and stimulate its kinase activity (lane 11). Together, these data indicate that there are two regions of Dbf4 that bind Cdc7 Figure3.—Individual mu-tations in the Dbf4 Zn finger cause sensitivity to stress. (A) Clustal align-ment of the Dbf4 Zn finger among nine orthologs with the budding yeast number-ing above. Sc (S. cerevisiae), Sp (Schizosaccharomyces pombe), Hs (Homo sapiens), Mm (Mus musculus), Xl (Xenopus laevis), Dm ( Dro-sophila melanogastor), An ( As-pergillus nidulans), Cg (Cricetulus griseus), and Ec (Equus caballus). Highly conserved residues are in red and identical residues are in blue. (B) The indi-cated mutants were spotted onto plates as in Figure 1. Wild-type Dbf4 and mutant proteins were expressed at similar levels (Figure S1B) (C) The dbf4-6

temperature-sensitive strain (M2400) was transformed with low- or high-copy vec-tors containing theDBF4or

and stimulate its kinase activity. The Zn-finger domain binds Cdc7 and is required for full stimulation of Cdc7 kinase activity, and a region spanning residues 312–418 also contributes to Cdc7 binding. This second Cdc7-binding region also includes residues from motif M and mediates the primary physical interaction since the Zn finger alone is not sufficient for Cdc7 binding. These results are summarized in Figure 5B.

To confirm whether the Dbf4 Zn finger and/or residues 312–418 interacted independently with Cdc7, we performed a two-hybrid analysis. Dbf4 residues 630– 704 (that included the Zn finger) interacted with Cdc7 by a two-hybrid assay (Figure 5C). This interaction absolutely required the conserved cysteine and histidine residues within the C2H2 Zn finger. Residues 252–418

also bound Cdc7, but residues 312–418 alone could not interact with Cdc7 (Figure 5D). We made several two-hybrid constructs spanning only motif M, but un-fortunately, these were not expressed. A Dbf4 BRCT two-hybrid protein was expressed, but this did not interact with Cdc7 (data not shown). In summary, the two-hybrid data are consistent with our mutational analysis and

together strongly suggest that Dbf4 uses its Zn finger and an extended 160-residue sequence, including motif M, to interact with Cdc7.

The Rev1 BRCT domain can substitute for the Dbf4 BRCT-like domain:In contrast to regions required for binding and activating Cdc7 kinase, a significant Dbf4 N-terminal region (residues 1–265) is largely dispens-able for its replication activity (Gabrielseet al.2006). The Dbf4 N terminus contains a destruction box, two classic NLSs, and residues that bind Cdc5 (Hardyand Pautz1996; Milleret al.2009) and Rad53 (Kihara et al. 2000; Duncker et al. 2002). In addition, Dbf4 contains a BRCT-like domain from residues117–218. Deletion of this motif results in slow growth, DNA damage sensitivity, and slowed progression through S-phase (Gabrielse et al. 2006). However, these phenotypes are likely due to a concomitant decrease in Dbf4 nuclear localization, since a dbf4-ND221 mu-tant that contains a heterologous NLS largely reverses these phenotypes with the exception that the dbf4-NLS-ND221 mutant retains a moderate sensitivity to HU.

Figure4.—Mutations in the Dbf4 Zn finger af-fect UV mutagenesis and cause slow S-phase pro-gression. (A) Wild-type DBF4 (M2040) or the indicated mutants dbf4-CC661,664AA (M2186),

dbf4-CD629 (M2183), dbf4-P277L (M3361), and

dbf4-D118-221 (M3362) were UV irradiated and tested for survival (inset) or Trp1 reversion events. (B) Wild type (M1016), dbf4-CD629

To further investigate whether Dbf4 encoded abona fideBRCT-like repeat, we performed domain-swapping experiments using known BRCT domains inS. cerevisiae to see if any could substitute for the Dbf4 BRCT-like domain. There are 11 proteins in yeast that contain single or tandem BRCT repeats (Table 2) (Borket al. 1997). Two of these proteins, Dbp11 and Esc4, contain two and three pairs of tandem BRCT repeats, respec-tively. We found that placing any tandem BRCT repeat on the N terminus of Dbf4 was lethal (Table 2 and Figure 6B). However, chimeric Dbf4 proteins contain-ing only a scontain-ingle BRCT domain suppressed the viability defect of thedbf4Dwith the exception of the Fcp1- and Chs5-Dbf4 chimeras (Table 2 and Figure 6B). A Western blot of all the Dbf4 chimeric proteins that rescued

viability showed that they were expressed very similarly to Dbf4-ND221 protein (Figure 6C). Interestingly, the Rev1 BRCT domain swap was unique in that it rescued all of the DNA damage sensitivity of the dbf4-ND221 mutant (Figure 6D), including its HU sensitivity. Rev1 is a member of the translesion DNA polymerase family, and its BRCT domain is required for this activity (Lawrence 2004). The mouse Rev1 BRCT domain can interact with PCNA by co-immunoprecipitation and contributes to its targeting to replication foci (Guoet al.2006), but the precise function of the yeast Rev1 BRCT domain is still unknown.

within the Rev1 BRCT domain at positions 202–218, we mutated one of the conserved lysines to methionine (K216M), disrupting the match to the NLS. The Rev1(K216M)-Dbf4 still suppressed the growth and DNA damage sensitivity of thedbf4-ND221mutant, suggesting that the suppression that we observed was not due to the putative Rev1 NLS (Figure 7, A and B). Furthermore, a dbf4-NLS-ND221 mutant retains HU sensitivity, but the Rev1-Dbf4 chimera has wild-type sensitivity to HU (Figure 6D). In contrast, suppression of thedbf4-ND221 pheno-types required the structural integrity of the Rev1 BRCT domain (Figure 7, A and B). Mutations of conserved BRCT family residues (see Borket al.1997) in the Rev1-Dbf4 chimera either entirely abolished Rev1-Dbf4 activity (G193L, G193R, G193V, W231A) or eliminated the DNA damage resistance (G193A, P229A, D230A, and W231L). The G193R substitution defines the originalrev1-1mutant that is defective for UV-induced mutagenesis (Lawrence 2004). The equivalent protein levels of representative Rev1-Dbf4 point mutants are shown in Figure 7C. Two Rev1 BRCT mutations that eliminated Dbf4 activity entirely (G193R and W231A) were not expressed (Figure S1C), indicating that they are destabilizing mutations. In conclusion, our data indicate that the Rev1 BRCT domain functionally substitutes for the Dbf4 BRCT-like domain, suggesting that Dbf4 and Rev1 interact with a shared protein. The identity of this protein remains unknown for both Dbf4 and Rev1, but an informed hypothesis suggests that it is a protein present at replication forks.

DISCUSSION

In this study, we mapped two regions on budding yeast that Dbf4 required for binding and activating Cdc7

kinase. One of these regions, motif C, likely encodes a functional Zn-finger domain on the basis of our point mutational analysis, and the other region includes motif M plus significant downstream sequences. We also presented further evidence that Dbf4 contains an N-terminal BRCT-like domain that could target the kinase to a replication fork protein. Finally, we showed that the Zn finger but not the BRCT-like domain is required for induced mutagenesis in yeast.

Deletion analysis of theS. pombeDfp1 (ScDbf4) subunit showed that two regions contributed to Hsk1 (ScCdc7) binding and kinase activation (Oginoet al. 2001; Fung et al.2002). In Dfp1, regions encompassing motif M or motif C bind independently to Hsk1 and contribute to kinase activation. In fact, a mutant that deleted the intervening residues between motif M and motif C could complement Dfp1 activity, strongly suggesting that the S. pombeDfp1 required only these two regions to bind and activate Hsk1in vivo. Deletion of motif C had little effect on Cdc7 kinase activity in one study (Oginoet al.2001) but had more pronounced effects in the second (Fung et al.2002) although this did not seem to affect S-phase progression. On the basis of further mutational data it was proposed that a redundancy existed between motifs N and C since motif N or motif C could be deleted but deletion of both motifs was not tolerated (Oginoet al. 2001).

Our mutational analysis of the S. cerevisiae Dbf4 is consistent with these earlier studies but reveals some important differences regarding the budding yeast subunit. We find that C-terminal mutants removing the Dbf4 Zn finger exhibit a significant defect in Cdc7 kinase activation, which is consistent with the significant defect that these mutants have entering and progressing TABLE 2

BRCT-Dbf4 chimeric proteins

Protein name

No. of BRCT repeats

No. of BRCT repeats in chimeraa

Rescue ofdbf4D

lethalityb

Suppression of DNA damage phenotype

Dbf4 1 1 1 1

Chs5 1 1 NA

Dnl4 2 2 NA

Dpb11 4 2 (aa1-228) NA

Dpb11 4 2 (aa306-592) NA

Esc4 6 0 NA NA

Fcp1 1 1 NA

Nop7 1 1 1

Pol4 1 1 1

Rad9 2 2 NA

Rap1 1 1 1

Rev1 1 1 1 1

Rfc1 1 1 1

a

BRCT-Dbf4 chimeras were constructed as indicated in Figure 6A and as described in materials and methods_.

b_{Chimeras were tested for the ability to suppress the}

through S-phase. A small 60-amino-acid region span-ning the Dfp1 motif M could interact with Hsk1 by co-IP and was auto-phosphorylated (Ogino et al. 2001). Although we were unable to detect binding of only motif M to Cdc7 for technical reasons, we found that the in vitro Dbf4-Cdc7 interaction required motif M plus 100 amino acids downstream of motif M. Our finding that a C-terminal deletion mutant to residue 312 (that retained motif M) exhibited a total loss of Cdc7 binding and kinase activation (although the mutant Dbf4 protein was abundantly expressed) supports this

conclusion. Furthermore, we previously showed that an N-terminal Dbf4 mutant that deletes most of motif M (ND292) retains the ability to bind and activate Cdc7 kinase in vitro, but a further deletion to residue 357 abolishes Cdc7 binding and kinase activation (Figure 5). Thus, both the budding and fission yeast Dbf4 subunits bind to Cdc7 kinase using a bipartite interac-tion. However, Dbf4 differs somewhat from Dfp1 in that motif M plus substantial downstream sequences com-pose the first binding site. The Zn finger comprises the second binding site as in Dfp1, but in budding yeast Figure 6.—The Rev1 BRCT domain suppresses loss of the Dbf4 BRCT-like sequence. (A) Heterologous BRCT domains were cloned on anNcoI fragment into the dbf4-ND221 gene. (B) M895 [dbf4D/pMW490 (DBF4 URA3)] was transformed with wild type (WT) DBF4, dbf4-ND221, or various chimeric dbf4-BRCT-ND221 plasmids (LEU2) and tested for the ability to rescue viability of dbf4D. (C) Western blot of total cell extracts showing wild-type Dbf4, Dbf4-ND221, and the viable BRCT-containing chimeric proteins. (D) Tenfold serial dilutions of wild type (M1016), dbf4-ND221 (M1173), and the various chimeric strains were spotted onto YPD (1/ ) and the indicated drugs and were incubated for 2 days at 30°.

the Zn finger contributes substantially to kinase activation.

Point mutations in the conserved C2H2 Zn-binding

residues of Dbf4 cause a substantial defect in cell growth and S-phase progression and also give rise to DNA damage and temperature sensitivities. The cell growth and DNA damage phenotypes likely result from a significant defect in activating Cdc7 kinase, which is required for the initiation of DNA replication. We pre-viously showed that increased MMS sensitivity is a common phenotype of replication initiation mutants (Gabrielse et al. 2006). Therefore, initiation mutants are MMS sensitive probably because too few replication origins are activated in the presence of MMS. Similarly, the bleomycin sensitivity ofdbf4N-terminal mutants was also accompanied by decreased initiation activity, a phenotype also shared by several other initiation mu-tants. Importantly, both the initiation defect and the bleomycin sensitivity of thedbf4-ND221 N-terminal mu-tant were rescued by adding back a functional NLS (Gabrielseet al.2006). Therefore, it seems possible that the MMS and bleomycin sensitivities of thedbf4Zn-finger mutants result from the initiation defect and that Cdc7-Dbf4 kinase is not directly involved in repair of alkylation damage or double-strand breaks. However, it is also pos-sible that Cdc7-Dbf4 operates in a direct pathway for repair of alkylation damage as suggested in fission yeast (Fung et al.2002; Sommarivaet al. 2005). For example, when Cdc7-Dbf4 promotes the initiation of DNA replication, this might be coupled with some Cdc7-Dbf4-dependent modification of the replication fork that enables cells to respond to particular types of DNA damage.

On the basis of multiple sequence alignments of Dbf4 orthologs and mutational data we previously suggested that Dbf4 encodes a BRCT-like domain that is dispensable for its essential mitotic activities (Gabrielseet al.2006). To further test this hypothesis, we performed domain-swapping experiments with known BRCT domains in yeast to see if we could rescue the dbf4-ND221mutant phenotypes using abona fideBRCT domain. None of the tandem BRCTrepeats in yeast even allowed viability when fused to the N terminus of Dbf4-ND221 protein;i.e., they inactivated the essential activity of Dbf4. This inviability might arise because the tandem BRCT domains target Cdc7 kinase away from its essential replication substrates to those phosphorylated substrates recognized by the respective BRCT domains. In contrast, we found that the single Rev1 BRCT domain suppressed all of the mutant phenotypes of dbf4-ND221 and that this suppression depended on conserved BRCT residues. This suggests that Dbf4 does encode a BRCT domain and that the Rev1 and Dbf4 domains interact with a common protein or protein structure. Indeed, a recent crystal structure of the Dbf4 BRCT motif confirms that it encodes a bona fide BRCT domain (A. Guarne, personal communication).

The C-terminal Dbf4 mutants are not sensitive to UV irradiation; however, they are almost completely

defective in UV-induced mutagenesis. This phenotype is similar to therev1-1mutant, which affects the Rev1 translesion DNA polymerase (Lawrence 2004). In other words, rev1-1 is not particularly sensitive to UV irradiation, but it is largely defective in UV-induced mutagenesis. Rev1 plays a minor role for the bulk of UV repair, which is instead accomplished by nucleotide excision repair or by the error-free TLS polymerase, Rad30(Polh) (Lawrence2004; Prakashet al.2005). In contrast to thedbf4C-terminal mutants, we found that the BRCT domain wasnotrequired for induced muta-genesis. Dbf4 mutants have not been previously ana-lyzed for their ability to support induced mutagenesis, butcdc7hypomorphs are largely defective in this process (Njagiand Kilbey1982a,b). Since Dbf4 is required for the mitotic activity of Cdc7 kinase, it was reasonable that dbf4hypomorphs would also affect induced mutagene-sis, but this had not been demonstrated.

Since Rev1 is responsible for the bulk of UV-induced mutagenesis in yeast, our data suggest that Dbf4 promotes a Rev1-dependent step in this process. The Dbf4 Zn finger is required for induced mutagenesis either because it stimulates Cdc7 kinase activity or because it also targets the Cdc7-Dbf4 to a protein required for induced mutagenesis. In addition, BRCT swapping data suggest that both Rev1 and Dbf4 might bind a common replication protein either during normal fork progression, when fork stalling occurs, or during G2when it is thought that nonrepairable lesions

are repaired by TLS polymerases (Lehmannand Fuchs 2006). This protein could be PCNA since the mouse Rev1 BRCT domain has been shown to mediate an interaction with PCNA by co-immunoprecipitation from cell extracts (Guoet al.2006). However, the target of the yeast Rev1 BRCT domain is unknown, and we have also not been able to detect a two-hybrid in-teraction between the budding yeast Rev1 or Dbf4 BRCT domains with PCNA or a PCNA–ubiquitin fusion protein (data not shown). Thus the binding partner for both BRCT domains remains uncertain. It will be im-portant to determine the exact binding partner for the Dbf4 BRCT domain and also to determine how Cdc7-Dbf4 kinase promotes induced mutagenesis in yeast.

We thank E. Craig and A. Sugino for yeast strains and the flow cytometry lab at the Van Andel Research Institute for technical support. We gratefully acknowledge the Van Andel Research Institute and a grant from the American Cancer Society (RSG0506301GMC), which supported this work.

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Supporting Information

http://www.genetics.org/cgi/content/full/genetics.109.110155/DC1

Budding Yeast Dbf4 Sequences Required for Cdc7 Kinase

Activation and Identification of a Functional Relationship Between

the Dbf4 and Rev1 BRCT Domains

Victoria Harkins, Carrie Gabrielse, Louise Haste and Michael Weinreich

Copyright © 2009 by the Genetics Society of America

V. Harkins et al.

2 SI




A

WT

C

!

654

C

!

629

C

!

598

C

!

575

C

!

511

*

*

*

*

_*

*

"

-Dbf4

B

WT

HH674,680LL

HH674,680AA

CC661,664AA

C664A

S677E

S677A

!

689-704

Dbf4

"

-Dbf4

Harkins et al. Fig S1

C

Rev1

BRCT

Rev1-G193R

Rev1-W231A

WT Dbf4

Dbf4-N

!

221 +Rev1 BRCT

"

-Dbf4










FIGURE S1.—Expression levels of Dbf4 proteins. (A) Whole cell extracts of the indicated mutants examined in Figure 1C were blotted for Dbf4 protein using a polyclonal antibody against

GST-Dbf4. Asterisks mark the Dbf4 proteins. (B) The mutants analyzed in Figure 3B were similarly probed for Dbf4 protein. (C) M895 strains (dbf4Δ/pMW490 (DBF4 URA3) expressing Dbf4,

Rev1-G193R-Dbf4, or Rev1-W231A-Dbf4 BRCT domain chimeras on plasmids were streak purified selecting for the REV1-DBF4 chimeric plasmid. Whole cell extracts were then probed for Dbf4 protein

V. Harkins et al. 3 SI




TABLE
S1


Plasmids





Name
 Description


pMW489
 pRS415‐DBF4
 pMW490
 pRS416‐DBF4
 pMW526
 pRS415
dbf4­NΔ65


pCG10
 pRS415
dbf4­NΔ109


pCG15
 pRS415
dbf4­Δ136­221


pCG29
 pRS415
dbf4­NΔ221


pCG45
 pCG29
x
RAD9
BRCT
 pCG46
 pCG29
x
DNL4
BRCT
 pCG48
 pCG29
x
DPB11
BRCT
#1/2
 pCG51
 pCG29
x
POL4
BRCT
 pCG54
 pCG29
x
REV1
BRCT
 pCG56
 pCG29
x
RAP1
BRCT
 pCG70
 pCG29
x
rev1­P229A
BRCT
 pCG71
 pCG29
x
rev1­D230A
BRCT
 pCG72
 pCG29
x
rev1­W231A
BRCT
 pCG73
 pCG29
x
rev1­G193R
BRCT
 pCG76
 pRS415
dbf4­NLS­NΔ221


pCG92
 pAcSG2
dbf4­NΔ292


pCG94
 pCG29
x
NOP7
BRCT
 pCG95
 pCG29
x
FCP1
BRCT
 pCG96
 pCG29
x
RFC1
BRCT


pCG119
 pCG29
x
rev1­GG192,193AA
BRCT
 pCG122
 pCG29
x
rev1­G193A
BRCT
 pCG126
 pRS415
dbf4­NLS­aa221­417
 pCG165
 pCG29
x
DPB11
BRCT
#3/4
 pCG175
 pGBKT7‐CDC7


pCG183
 pAcSG2
dbf4­NΔ357


pCG193
 pAcPK30
dbf4­Δ312­418


pCG209
 pGAD
dbf4­aa311­418


pCG211
 pAcSG2
dbf4­NΔ292,
CC661,684AA


V. Harkins et al.

4 SI




pLVH51
 pAcPK30
dbf4­CC661,664AA
 pLVH52
 pAcPK30
dbf4­CΔ629


pLVH53
 pRS425‐CDC7
 pLVH55
 pRS415
dbf4­S677A
 pLVH56
 pRS415
dbf4­C664A
 pLVH58
 pRS415
dbf4­CΔ689


pLVH59
 pRS415
dbf4­S677E
 pLVH60
 pRS415
dbf4­HH674,680AA
 pLVH62
 pLVH47
HH674,680LL
 pLVH63
 pLVH47
CC661,664AA
 pLVH64
 pLVH47
C661A


pVMH3
 pRS415
dbf4­CC661,664AA
 pVMH5
 pRS415
dbf4­HH674,680LL
 pVMH7
 pRS415
dbf4­CΔ654


pVMH14
 pCG29
x
CHS5
BRCT
 pVMH16
 pRS415
dbf4­CΔ418


pVMH18
 pRS415
dbf4­CΔ511


pVMH21
 pRS415
dbf4­CΔ575


pVMH22
 pRS415
dbf4­CΔ598


pVMH23
 pRS415
dbf4­CΔ629


pVMH59
 pRS415
dbf4­CΔ312


pVMH61
 pRS415
dbf4­CΔ357


pVMH71
 pCG76
x
CΔ629


pVMH72
 pCG76
x
CΔ654


pVMH79
 pCG76
x
CC661,664AA
 pVMH81
 pAcPK30
x
dbf4­CΔ418


pVMH84
 pAcPK30
x
dbf4­CΔ312


pVMH85
 pCG10
x
CΔ654


pVMH86
 pCG10
x
CΔ629


pVMH87
 pCG10
x
CC661,664AA
 pVMH91
 pMW526
x
CΔ654


pVMH93
 pMW526
x
CΔ629


pVMH95
 pMW526
x
CC661,664AA
 pVMH97
 pRS415
dbf4­P277L
 pVMH101
 pMW526
x
CΔ418


pVMH107
 pVMH97
x
CΔ654


pVMH109
 pVMH97
x
CΔ629


pVMH110
 pVMH97
x
CΔ418


pVMH112
 pVMH97
x
CC661,664AA
 pVMH121
 pCG15
x
CC661,664AA
 pVMH123
 pCG15
x
CΔ654


V. Harkins et al. 5 SI




pVMH127
 pCG15
x
CΔ629


pVMH130
 pRS415
dbf4­Δ118­221


pVMH131
 pVMH130
x
CC661,664AA
 pVMH132
 pVMH130
x
CΔ654


pVMH133
 pVMH130
x
CΔ418


pVMH134
 pVMH130
x
CΔ629


pVMH136
 pRS415
dbf4­Δ122­221