H om ologous recom bination

There are several distinct phases of HR repair of DNA DSBs (figure 1-10). The double-

lonising radiation

5'-to-3' trimming Pairing with template

Strand Invasion

1

I

DNA synthesis

Resolution Ligation

Figure 1-10 General outline for the fundamental steps of Homologous Recombination

repair of DNA DSBs generated by ionising irradiation.

Stranded ends of the DNA DSB are first processed to generate 3’ single-stranded overhangs that first invades and base-pairs with a homologous DNA molecule, preferentially a sister chromatid. The 3’ single-stranded overhangs are then used as primers for DNA synthesis to regenerate the missing sequence of the damaged strand. Once, DNA synthesis is complete the two DNA molecules are unwound and separated by a process known as resolution, followed by ligation to restore the two DNA helices. The process of homologous recombination is also utilized in recombination during meiosis and as a backup mechanism that repairs errors encountered during DNA replication (reviewed in Haber, 2000a; Haber, 2000b; Marians, 2000).

Although the general process of HR that repairs DNA DSBs generated by ionising irradiation is fairly well established, less is known about its components, their function and regulation. Our current understanding of the mechanisms of HR is drawn from initial work using the budding yeast S. cerevisiae, and more recent work using mammalian cells. Based on these studies the following model of mammalian HR can be established. The components of HR are summarised in table 1-4.

Human

S. cerevisiae RadSI R a d S I p R adSIB R adSIC Rad51D KRCC2 KRCC3 Rad 5 2 R a d 5 2 p Rad54 R a d 5 4 p RadSSp R a d 5 7 p RPR RPR R adSe RadSBp M r e l 1 M r e l I p N b sl Hrs2p B r c a l B rca2

Table 1-4 Components of human and S. cerevisiae homologous recombination.

When the DNA DSB is generated, a resection process is initiated to generate 3’ single­ stranded overhangs (Sugawara, et al., 1992). This is an important step for several reasons. The 3’ single-stranded overhangs are used to find a suitable template to which they first pair up with (synapse) and then invade (strand invasion). The process of strand invasion results in base pairing of the template molecule with the 3’ single-stranded overhang whose 3 -OH group is subsequently used as a substrate for DNA synthesis. The generation of the 3’ single-stranded overhangs from the double-stranded ends is

presumably carried out by nucleases possibly acting in concert with DNA helicases. The identities of these proteins are not known, but there is evidence that the budding yeast S. cerevisiae protein complex Mrel Ip-Rad50p-Xrs2 (Mrel 1-Rad50-Nbsl in mammals) is an important component in this process (Bressan, et al., 1999; Ivanov, et al., 1994; Lee, et al., 1998). The exact role of this complex is not understood. Mrel 1 is a 3’ to 5 ’

exonuclease whose activity is not required for resection (Bressan, et al., 1999; Moreau, et al., 1999; Pauli, et al., 1998). Rather, this protein complex might function as a regulator of the exonucleases and/or helicases that are required for resection.

Once the 3 ’ single-stranded overhangs have been generated, a number of proteins act in concert to first synapse and then facilitate the strand invasion of the 3 ’ single-stranded overhang with a suitable template. Strand invasion has been performed in vitro using human proteins and requires the R adSl, Rad52 and the single-strand binding RPA proteins (Mcllwraith, et al., 2000). Although these three proteins can catalyse strand invasion in vitro, the same mechanism in vivo requires additional factors.

The central factor in these early phases o f HR is R adSl, which forms protein filaments along the 3 ’ single-stranded overhang, and plays important roles by first pairing the 3 ’ single-stranded overhang with the template molecule and then catalysing the subsequent strand invasion (Baumann, e ta l., 1997; Sung, 1994; Sung, e ta l., 1995).

Several proteins assist RadSl in synapsing and strand invasion. The RPA protein binds to the 3 ’ single-stranded overhang and prevents secondary structures from forming that might inhibit downstream repair events (Baumann, et al., 1997; Sugiyama, et al., 1997). A side effect of RPA binding to the overhang that needs to be overcome is that it hinders RadSl from interacting with the single-stranded DNA (Sung, 1997a). There is evidence from both yeast and human cells that the RadS2 protein, which in humans is able to bind to both single-stranded and double-stranded ends of DNA, overcomes this inhibitory effect, possibly by direct protein interaction with RadSl (Benson, eta l., 1998; New, et al., 1998; Parsons, et al., 2(XX); Shinohara, et al., 1998; Sung, 1997a; Van Dyck, et al.,

1999). Therefore, it is plausible that once resection is complete, RadS2 binds to the ends, whilst RPA binds along the 3 ’ single-stranded overhangs. Once these initial interactions are complete, RadS2 then recruits and facilitates RadSl binding to the overhang.

Mice that are null for RadS2 are viable and, although having reduced HR, are not noticeable sensitive to ionising irradiation, suggesting that there are other proteins involved in the process o f facilitating RadSl binding to the 3 ’ single-stranded overhang (Rijkers, et al., 1998). Two such candidates in S. cerevisiae are two RadSl like proteins, RadSSp and RadS7p, that form a heterodimer, which has been shown to assist RadSl binding to the overhang (Sung, 1997b). Recent studies have demonstrated that there are several mammalian RadSl like proteins. These proteins are XRCC2, XRCC3, RadSIB, R adSlC and RadSlD (Albala, et al., 1997; Cartwright, et al., 1998a; Cartwright, et al.,

1998b; Dosanjh, e ta l., 1998; Liu, e ta l., 1998b; Pittman, e ta l., 1998; Rice, e ta l., 1997; Tambini, et al., 1997). These RadSl paralogs are implicated in HR but their exact roles

in this process are not well understood (Takata, et al., 2(X)1). There is, however, evidence that several of the RadsSl paralogs are capable of interacting with other RadSl paralogs and RadSl itself (table 1-S), suggesting that these proteins might have functions in the early phases of HR including facilitating RadSl binding to the overhang (Braybrooke, et al., 2(XX); Dosanjh, et al., 1998; Kurumizaka, et al., 2(X)1; Liu, et al., 1998b; Masson, et al., 2001; O'Regan, et al., 2001; Schild, e ta l., 2000).

Name

Interacts with

R a d S l R adS IB R adS IC R adS lD KRCC2 KRCC3 RRCC2, RRCC3 RadSIC R adSIB , R adSIB , HRCC3 R adSIC , KRCC2 R a d S l , R adSIB R a d S l, R adSIC

Table 1-5 Interactions between Rad51 paralogs (see text for references).

Another protein that is implicated in HR is RadS4. Mice and chicken cells that harbour targeted disruptions of the RadS4 gene are characterised by hypersensitivity to ionising irradiation and compromised HR, suggesting that Rad54 plays an important role in HR (Bezzubova, et al., 1997; Essers, et al., 1997). There is evidence suggesting that Rad54 interacts with the RadSl protein filament on the 3’ single-stranded overhang and then assists RadSl in pairing the overhang with the template molecule (Mazin, et al.,2000; Petukhova, et al., 1998; Tan, et al., 1999).

Two other proteins that might be involved in the early phases of HR are the tumour suppressor genes Brcal and Brca2. Cells that are deficient for Brcal and Brca2 are sensitive to ionising irradiation and have compromised HR suggesting a role for these proteins in this repair process (Gowen, et al., 1998; Moynahan, et al., 1999; Moynahan, et al., 2001; Sharan, et al., 1997). The function of Brcal and Brca2 in HR might be

mediated via interactions with Mrel 1-RadSO-Nbsl and RadSl proteins. Brcal interacts directly with RadSO and is found in the same foci as Mrel 1-RadSO-Nbsl following treatment with ionising irradiation, and both Brcal and Brca2 associate with RadSl in vivo (Chen, et al., 1998a; Scully, et al., 1997; Zhong, et al., 1999). This interaction appears to be direct for Brca2 and indirect for Brcal (Chen, et al., 1998b; Sharan, et al.,

suggest that the association between R adSl, B rcal and Brca2 occurs via direct binding of RadSl to Brca2 and that B rcal associates with RadSl via its interaction with Brca2 (Chen, et al., 1998a).

Once the pairing is complete, RadSl catalyses the strand invasion (Sung, 1994).

Following this process DNA synthesis is initiated to restore the missing DNA sequences. The elongation, or branch migration, of the two 3’ single-stranded overhangs in opposite directions along the double-stranded template molecule generates two Holliday junctions. When the DNA synthesis has regenerated enough DNA to restore the damaged DNA molecule, the newly synthesized strands are unwound from the template DNA and ligated to the damaged DNA thereby restoring the DNA helix. In the prokaryote E. coli the RuvA and RuvB proteins are involved in branch migration whereas the RuvC protein performs the resolution of Holliday junctions (reviewed in West, 1996). These proteins are also capable of performing branch migration and resolution in vitro (Eggleston, et al.,

1997). Recently, a mammalian protein complex was identified, which is able to perform both branch migration and resolution of Holliday junctions in a manner that is

homologous to the RuvABC system of E. coli (Constantinou, et al., 2001). Further work is required to establish the identities of the components of this complex, and to investigate whether it is required for HR.

There is evidence that proteins that are involved in the DNA damage response pathway might regulate HR by means of RadSl. One mechanism by which this regulation might take place is through the protein kinases ATM and c-Abl. In response to ionising irradiation, the ATM protein interacts with, and activates c-Abl (Baskaran, et al., 1997; Shafman, et al., 1997). Once activated, c-Abl phosphorylates R adSl, a modification that enhances RadSl-RadS2 interaction (Chen, et al., 1999). The RadSl and RadS2

interaction following treatment with ionising irradiation is impaired in both ATM and c- Abl deficient cells, and that in human cells the ATM protein, c-Abl and RadSl can be co- immunoprecipitated (Chen, et al., 1999). These findings suggest that following DNA damage by ionising irradiation, the ATM protein activates c-Abl, which, in turn,

facilitates RadSl-RadS2 interaction by phosphorylating RadSl. The ATM protein is also reported to regulate HR through phosphorylation of B rcal (Cortez, et al., 1999). This observation is based on the observations that B rcal, which is a substrate of the ATM protein is not phosphorylated in cells that do not have functional ATM protein, and that

expression of B rcal protein lacking the ATM recognition m otif in cells that are deficient for B rcal fails to revert their ionising irradiation sensitive phenotype.

In document An investigation of genes involved in doubled-stranded break repair of DNA (Page 42-47)