Post-translational modification of factors involved in homologous recombination☆,☆☆
Introduction
While it is common knowledge that exogenous factors such as genotoxic chemicals and ionising radiation (IR) can cause DNA damage, it is also important to acknowledge that DNA damage can arise as a consequence of routine endogenous processes. Many forms of DNA damage can take place and cells are armed with an arsenal of DNA repair mechanisms to counteract the devastating effects of these genome destabilising events. A particularly severe form of DNA damage is a DNA double-strand break (DSB), which divides a single normal chromosome into two pathological chromosomes. Accordingly, DSBs are recognised as strong drivers of cell death and tumorigenesis [1,2]. A DSB poses a unique challenge to the cell because it introduces a discontinuity in the DNA molecule, eliminating the possibility of simply utilising the information encoded in the complementary strand to restore and repair the damage, a strategy commonly exploited by several DNA repair mechanisms. Homologous recombination (HR) is a DSB repair mechanism that identifies another region within the genome that shares high sequence similarity (i.e., homology) with the DSB site, and allows this homologous region to be used as a template for DSB repair. This ability of HR to identify homologous sequences is also exploited in meiosis, where it promotes the correct pairing of homologous chromosomes (i.e., cognate chromosomes of different parental origin) and their faithful segregation [3]. Thus, HR plays a central role in two critical aspects of an organism’s lifecycle: growth and reproduction.
HR is initiated when the DNA ends at a DSB are subjected to nucleolytic processing that exposes 3′-ended single-stranded DNA (ssDNA) overhangs (Fig. 1) [4]. These ssDNA tracts are first bound by the ssDNA-binding protein RPA, then by the RecA-family recombinase Rad51, which forms a right-handed helical filament on the ssDNA [5]. This Rad51-ssDNA nucleoprotein filament, which is often referred to as the presynaptic filament, is capable of interrogating the genome for regions that are similar in sequence to the filamentous ssDNA in a process known as the homology search [6]. Once homology is located, the Rad51-ssDNA filament invades into the intact duplex DNA and forms base pairs with the complementary strand of the duplex, effectively displacing the non-complementary strand and forming a displacement loop (D-loop) structure. This process is known as DNA strand exchange. Importantly, the 3′-end of the invading strand can then be utilised by DNA polymerases to extend the invading strand according to the paired complementary strand. Typically, the invading strand then dissociates from the duplex, and having been extended by DNA synthesis, can anneal to the ssDNA on the other side of the DSB [7]. Following further gap filling and ligation of ends, DSB repair by HR is complete. While this synthesis-dependent strand annealing (SDSA) is one form of HR-mediated DSB repair and exclusively yields non-crossover outcomes, there are also more complicated possibilities that can yield crossover outcomes (Fig. 1) [8].
In the budding yeast Saccharomyces cerevisiae, the predominant mechanism giving rise to crossovers involves incorporation of the second end of the DSB into the D-loop structure via a process termed second-end capture. This leads to the formation of a double Holliday junction, an iconic structure in the field of HR [9]. Holliday junction dissolution by helicase and type IA topoisomerase enzymes exclusively yields non-crossovers, whereas Holliday junction resolution by structure-specific endonucleases known as resolvases can yield either non-crossover or crossover outcomes [10]. This is known as the double-strand break repair (DSBR) model of HR. This mode of crossover formation is relatively rare in the fission yeast Schizosaccharomyces pombe, where the primary mode of crossover formation involves cleavage of the D-loop structure, leading to the establishment of a single Holliday junction [11]. As with the double Holliday junction, the single Holliday junction can be cleaved to yield either non-crossover or crossover outcomes [9,12]. Both the SDSA and the DSBR models are depicted in Fig. 1. Even more variables are introduced in diploid cells, where homologous DNA is present in the form of the sister chromatid and the homologous chromosome. Utilisation of the sister chromatid in a manner that results in non-crossovers is favoured in mitotic (i.e., somatic) cells, whereas a specialised mode of HR that preferentially engages homologous chromosomes and promotes crossover outcomes operates in meiotic (i.e., germ line) cells [3].
This molecular model places Rad51 at the heart of HR. Consistently, yeast strains lacking Rad51 (denoted as rad51Δ) show a slow-growth phenotype and are severely sensitive to DNA damaging agents [13,14], whereas mice lacking Rad51 display embryonic lethality [15], highlighting the essentiality of HR in higher eukaryotes. However, it is important to note that Rad51 requires the help of several other proteins to fulfil its function. These proteins, known as recombination auxiliary factors (RAFs hereafter), promote HR by physically interacting with Rad51 and potentiating its activity. It is becoming increasingly obvious that, along with Rad51, many of these RAFs are subjected to post-translational modifications (PTMs). These PTMs are particularly pertinent to the regulation of HR since HR proficiency oscillates with the mitotic cell cycle. HR is repressed during the G1 phase and stimulated during the S and G2 phases, where replication has generated sister chromatids (i.e., identical templates) that can be utilised for accurate DSB repair. By contrast, more error-prone DSB repair mechanisms such as non-homologous end joining are utilised primarily in G1 in yeast cells, and more generally throughout the cell cycle in higher eukaryotes [16].
Rather than provide a catalogue of every known PTM of every HR factor, this review aims to provide a summary of how PTMs regulate the ability of RAFs to promote recombinational DNA repair. The PTMs discussed here are summarised in Table 1; this is not an exhaustive list but is provided in the hope that it will be a useful resource to accompany this article. The primary focus of this review will be on mitotic cells but relevant PTMs in meiotic cells will also be mentioned. Although we will discuss the PTMs of Rad51 that are thought to affect its activity, as well as the modifications of the eukaryotic ssDNA-binding protein Replication Protein A (RPA), the bulk of this review will focus on RAFs. We will describe the cases where a particular PTM has been experimentally shown to affect Rad51 modulation by RAFs, with the aim of highlighting the diverse ways in which RAFs are regulated by PTMs. We will also mention more speculative cases where a function for the PTM can only be inferred. Finally, we will discuss why such regulation of RAF function might be necessary.
Section snippets
An overview of Rad51 PTMs
Given that Rad51 is the central player in HR, it is worthwhile to include a discussion of the post-translational regulation of Rad51. Here, we will briefly mention the PTMs of Rad51 to demonstrate that, in addition to RAFs, it too is subjected to extensive regulation at the post-translational level. For readers seeking a more focused discussion specifically about Rad51, we recommend recent reviews that may be of interest [17,18].
The first PTM of RAD51 (note that the nomenclature for human
RPA: an extensively modified complex that is essential for HR
RPA is a heterotrimeric complex consisting of Rfa1, Rfa2, and Rfa3, which are the large (∼70 kDa), intermediate (∼30 kDa), and small (∼14 kDa) subunits, respectively. In addition to its critical role in signalling DNA damage [39,40], RPA participates in all forms of DNA metabolism where ssDNA is exposed, including DNA replication and HR [41]. RPA binds ssDNA with high affinity and stabilises it by preventing secondary structure formation and the spontaneous re-annealing of complementary
Phosphorylation of the Rad51 paralogues Rad55, XRCC2, and XRCC3
Rad55 and Rad57 are paralogues of Rad51 in yeast and the three proteins share high sequence similarity across their ATPase core domains [18]. Biochemical reconstitutions with S. cerevisiae proteins suggested that Rad55 and Rad57 form an obligate heterodimer that interacts with Rad51 and functions in an early step of HR by promoting the formation of the Rad51-ssDNA nucleoprotein filament [[68], [69], [70]]. This notion is supported by cytological experiments in both S. cerevisiae and S. pombe
SUMOylation and phosphorylation of Rad52
Rad52 is the prototypical RAF in yeast and has been shown to mediate Rad51 filament formation on RPA-coated ssDNA [[91], [92], [93], [94], [95], [96]]. Rad52 is known to be regulated by multiple PTMs. Experiments in S. pombe first demonstrated that Rad52 is SUMOylated [97]. Subsequent analysis in S. cerevisiae revealed that Rad52 SUMOylation is heavily induced by DNA damage and that mutation of Lys10, Lys11, and Lys220 to Arg essentially abrogates this modification [98]. This report suggested
The diverse post-translational landscape of Rad54
As a member of the Swi2/Snf2-family of dsDNA translocases, Rad54 is unique among RAFs in that it possesses a robust enzymatic activity (ATP hydrolysis) and is primarily involved in the later stages of HR [106,107]. Rad54 not only promotes Rad51-driven DNA strand exchange, but it also dissociates Rad51 from synaptic and postsynaptic complexes to promote polymerase-dependent DNA synthesis and completion of HR [[108], [109], [110], [111], [112]]. Furthermore, Rad54 shows the highest degree of
BRCA2, BRCA1-BARD1, and implications for human health
Rad52 is the major RAF that promotes Rad51 nucleation on RPA-coated ssDNA in S. cerevisiae and S. pombe. Rad52 also anneals complementary ssDNA in the later stages of HR and in Rad51-independent recombination events such as single-strand annealing [8]. However, there is a divergence of function in higher eukaryotes. The tumour suppressor protein BRCA2 promotes RAD51-ssDNA nucleoprotein filament formation but is devoid of ssDNA annealing activity, whereas RAD52 retains ssDNA annealing activity
Post-translational regulation of RAFs during meiosis
As mentioned earlier, a specialised mode of HR operates in meiotic cells, where interhomologue engagement is favoured over intersister engagement and crossover outcomes are promoted in order to achieve the reductional cell division and generate the genetic diversity that typify meiosis (Fig. 1) [3]. HR is highly induced in meiosis through the programmed induction of DSBs by Spo11, a type II topoisomerase-like protein. In addition to Rad51, many eukaryotes possess a meiosis-specific RecA-family
Putative regulatory functions for the phosphorylation of Swi5-Sfr1 and RAD51AP1
Swi5-Sfr1 is a RAF first identified in S. pombe as functioning in a sub-pathway of Rad51-dependent DNA repair [180]. Since then, orthologues of Swi5-Sfr1 have also been shown to promote HR in S. cerevisiae and mammals [[181], [182], [183], [184]]. Biochemical reconstitutions have demonstrated that SpSwi5-Sfr1 promotes HR by stabilising Rad51-ssDNA filaments and stimulating the ATPase activity of Rad51 [96,[185], [186], [187]]. Similar observations have been made for the S. cerevisiae homologue
Conclusions and perspectives
The mechanisms underlying Rad51 potentiation by RAFs are just starting to emerge. However, it has become increasingly clear in the last two decades that Rad51 and RAFs are regulated at the post-translational level. It has proven challenging to understand how these PTMs affect RAF function. A common theme that is relevant to BRCA2, ScRad54, and potentially SpSwi5-Sfr1 and RAD51AP1, is that phosphorylation of RAFs influences their physical interaction with Rad51. Acetylation of HsRAD54 also
Author contributions
B.A. prepared the manuscript with input from H.I. and H.T.
Declaration of Competing Interest
The authors have no conflicts of interest to declare.
Acknowledgements
We thank Xiaodong Zhang, Fumiko Esashi, and members of the Iwasaki laboratory for discussions. We also offer our sincere apologies to colleagues whose work was not discussed due to space constraints. Research in the Iwasaki laboratory is funded in part by Grants-in-Aid for Scientific Research (A) (JP18H03985 to H.I.), for Scientific Research (B) (JP18H02371 to H.T.), and for Early-Career Scientists (JP20K15713 to B.A.) from the Japan Society for the Promotion of Science (JSPS).
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This article is part of the special issue Maintenance of Active Genome Integrity.
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This Special Issue is edited by Sukesh R. Bhaumik.