Replication protein A

The use of genetic information encoded in DNA, requires unwinding of duplex DNA and exposure of single stranded DNA (ssDNA). In this state DNA is highly vulnerable to damage, as well as to the formation of spontaneous duplex DNA. ssDNA binding proteins exist in bacteria, archaea and eukaryotes, and all contain the oligonucleotide binding (OB) fold for ssDNA binding and oligomerisation (Flynn and Zou, 2010).

RPA is the primary eukaryotic ssDNA binding protein. It is required for all aspects of DNA metabolism that involves a ssDNA intermediate. These include DNA replication, recombination, repair, telomere maintenance and DNA damage response. RPA is a heterotrimer of 70, 32 and 14kDa subunits (RPA70, RPA32 and RPA14, respectively) that are encoded by three separate genes (Wold, 1997). RPA70 is the major DNA binding subunit and has four domains (Figure 3.1). The domains include an N-terminal regulatory, protein–protein interaction domain (amino acids 1–110, DBD-F) and three DNA binding domains (DBD): DBD-A (amino acids 181–290),

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DBD-B (amino acids 301–422), and DBD-C (amino acids 436–616) (Liu and Huang, 2016). RPA32 contains the DBD-D domain and DBD-E is present in RPA14 (Brill and Bastin-Shanower, 1998). The trimerisation occurs through interactions between domains DBD-C, D and E (Bochkareva et al., 2002). RPA32 contains an additional C-terminal winged helix domain for RPA interaction with other proteins and an N-terminal domain which becomes phosphorylated after DNA damage (Binz et al., 2004; Oakley and Patrick, 2010). DBD-E has a very weak interaction with DNA and mainly participates in trimerisation (Bochkarev et al., 1999).

Figure 3.1: Structure and binding of RPA (Chen and Wold, 2014)

(A) RPA binding to ssDNA by DNA binding domains (DBD) A-F, also showing the winged helix domain (wh) and the phosphorylation domain (Pd). Longer ssDNA binding (30nt) involves DBD-D. (B) The structure of Ustilago maydis RPA bound to ssDNA (black).

RPA1 (RPA70, green), RPA2 (RPA32, blue) and RPA3 (RPA14, red) (Fan and Pavletich, 2012).

RPA binds ssDNA 1000-fold more efficiently (nM affinity) than to dsDNA. It binds preferentially to longer ssDNA sequences and prefers polypyrimidine sequences to polypurines (Kim et al., 1994). The proposed dynamic binding mechanism involves an initial weak binding step by DBD-A and DBD-B to an 10-20 nucleotide segment.

Subsequent conformational changes allow binding of DBD-C, which in turn permits DBD-D access for binding to ssDNA substrates of 28-30 nucleotides (Bastin-Shanower and Brill, 2001).

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The long linker connecting the DBD-F protein interaction and DNA binding DBD-A domains confers some structural and dynamic autonomy on these functions and binding of ssDNA does not affect the protein interactions of RPA (Brosey et al., 2015).

The DNA binding domains (A and B) are connected by a short linker reflecting the high degree of coordination required for the initial binding of ssDNA. The combination increases binding affinity by around a 100-fold compared to each separate domain.

The binding of RPA to DNA appears to depend on free RPA concentration. It was shown that Saccharomyces cerevisiae RPA remains tightly associated with ssDNA in the absence of free RPA (Gibb et al., 2014). When free RPA becomes present in solution, RPA undergoes dissociation from DNA and exhibits rapid exchange between free and bound RPA, which may occur through a partially dissociated intermediate. This may allow other proteins to replace RPA. Gourdin et al. have shown that RPA exhibits differential binding to ssDNA under different conditions.

They speculate that transient RPA binding to DNA during replication and the pre-incision steps of RPA prevents checkpoint activation (Gourdin et al., 2014). RPA binding during replication stress or in the post-incision steps of NER is more stable and can activate ATR (ataxia telangiectasia and Rad3 related) and ATRIP (ATR-interacting protein).

A critical step of NER involves RPA70 binding the N-terminal region of XPA (xeroderma pigmentosum group A), while RPA32 binds its central region (Saijo et al., 2011). These interactions allow the recruitment of the XPF-ERCC1 and XPG endonucleases. XPG interacts with RPA directly (Saijo et al., 1996). RPA interacts with uracil-DNA glycosylase (UNG) and also facilitates long patch BER (DeMott et al., 1998; Nagelhus et al., 1997). Exonuclease 1-mediated excision during MMR is stimulated by RPA, which also protects the ssDNA generated by the excision (Genschel and Modrich, 2003). RPA has an important role in protecting ssDNA and removing secondary structures formed by ssDNA in homologous recombination (HR) and interacts with RAD51 and 52 (San Filippo et al., 2008).

Some genotoxic treatments may disrupt the interaction between RPA and XPA that is required for NER (Jiang et al., 2012). This disruption can occur through RPA32 hyperphosphorylation, which can promote the interaction of RPA with other factors

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(Deng et al., 2009; Shi et al., 2010). The association of RPA with ssDNA is more favourable than its binding to XPA and there is some overlap in the XPA interaction site and the DNA binding site of RPA70 (Daughdrill et al., 2003). ssDNA at collapsed DNA replication forks will sequester RPA and prevent it from binding XPA. The presence of a common surface of RPA involved in interacting with UNG2 (BER), Rad52 (HR) and XPA also results in competition between the different repair pathways for RPA (Mer et al., 2000). In the case of ultraviolet radiation (UV) and NER, it is proposed that at low DNA damage levels and few double strand breaks (DSBs), RPA associates with XPA for NER. At higher levels of DNA damage, DSBs at stalled DNA replication forks will compete with XPA, potentially aided by RPA hyperphosphorylation and sequester RPA (Wu et al., 2005). Consistent with this model, in the absence of translesion polymerase Polζ or in cells treated with hydroxyurea, persistently arrested replication forks sequester RPA (Tsaalbi-Shtylik et al., 2014). This is accompanied by inhibition of NER in trans. Therefore, free RPA levels have a significant regulatory role in the cell. Persistent fork stalling and the absence of free RPA can result in cell death (Tsaalbi-Shtylik et al., 2014).

Loss of any of the RPA subunits is lethal and non-lethal mutations result in genomic instability (Chen and Wold, 2014). Depletion of RPA can result in spontaneous DNA damage and G2/M checkpoint activation through ATM (ataxia telangiectasia mutated) (Halliwell, 1991; Haring et al., 2010; Santocanale et al., 1995). It has also been shown that depletion of RPA70 results in genome instability, activates the Fanconi anaemia (FA) pathway through chromatin association of the FA core complex and causes FANCD2 ubiquitination through ATR (Jang et al., 2016).

Haploinsufficiency of RPA in mice results in lymphoid tumours and shortened life span (Hass et al., 2010; Wang et al., 2005).

Replication fork stalling during replication stress increases the levels of exposed ssDNA. Under normal conditions, RPA stabilisation of stalled forks and activation of ATR, resolves the stalled fork and allows continuation of replication. Toledo et al.

showed that replication stress-induced depletion of RPA can cause exposed ssDNA to convert to DSBs (Toledo et al., 2013). This results in replication catastrophe and cell death. Overexpression of RPA enables cells to tolerate higher levels of replication stress. The activation of ATR by RPA and cell cycle arrest prevents new

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origin activation and therefore inhibits further generation of ssDNA to alleviate RPA exhaustion.