Structural basis of homologous recombination

Abstract

Homologous recombination (HR) is a pathway to faithfully repair DNA double-strand breaks (DSBs). At the core of this pathway is a DNA recombinase, which, as a nucleoprotein filament on ssDNA, pairs with homologous DNA as a template to repair the damaged site. In eukaryotes Rad51 is the recombinase capable of carrying out essential steps including strand invasion, homology search on the sister chromatid and strand exchange. Importantly, a tightly regulated process involving many protein factors has evolved to ensure proper localisation of this DNA repair machinery and its correct timing within the cell cycle. Dysregulation of any of the proteins involved can result in unchecked DNA damage, leading to uncontrolled cell division and cancer. Indeed, many are tumour suppressors and are key targets in the development of new cancer therapies. Over the past 40 years, our structural and mechanistic understanding of homologous recombination has steadily increased with notable recent advancements due to the advances in single particle cryo electron microscopy. These have resulted in higher resolution structural models of the signalling proteins ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad3-related protein), along with various structures of Rad51. However, structural information of the other major players involved, such as BRCA1 (breast cancer type 1 susceptibility protein) and BRCA2 (breast cancer type 2 susceptibility protein), has been limited to crystal structures of isolated domains and low-resolution electron microscopy reconstructions of the full-length proteins. Here we summarise the current structural understanding of homologous recombination, focusing on key proteins in recruitment and signalling events as well as the mediators for the Rad51 recombinase. Keywords Homologous recombination • Cryo electron microscopy • X-ray crystallography • Double-strand break repair • DNA damage signalling and repair Abbreviations ADP Adenosine diphosphate AMP-PNP Adenylyl-imidodiphosphate ATM Ataxia telangiectasia mutated ATP Adenosine triphosphate ATR Ataxia telangiectasia and Rad3-related protein BARD1 BRCA1-associated RING domain protein 1 BRCA1 Breast cancer type 1 susceptibility protein BRCA2 Breast cancer type 2 susceptibility protein BRCT BRCA1 C-terminal domain cryoEM Cryo electron microscopy DDR DNA damage response DSB Double-strand break EJ End joining MRE11 Meiotic recombination 11 homolog 1 MRN MRE11 RAD50 NBS1 NBS1 Nijmegen breakage syndrome protein 1 OB Oligonucleotide/oligosaccharide-binding PALB2 Partner and localiser of BRCA2 RPA Replication protein A

E2f5 is a versatile transcriptional activator required for spermatogenesis and multiciliated cell differentiation in zebrafish

“…We found 1352 transcripts upregulated in e2f5 mutants compared to wild-type, whereas 763 transcripts were downregulated (FDR< = 0.05). Surprisingly, most genes associated with homologous recombination, including rad51, blm, ino80 and brca2 [37,38], showed grossly normal expression levels in the mutants (Fig 3B, S2A Fig). However, the expression of dmc1, an essential gene involved in meiosis homologous recombination, was significantly downregulated in the mutants (Fig 3B, S2A Fig).…”

Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea

“…The L93-5 HL 647 oligonucleotide (5 HiLyte TM Fluor 647-AAA-GGC-GGT-AAT-ACG-GTT-ATC -CAC-AGA-ATC-AGG-GGA-TAA-CGC-AGG-AAA-GAA-CAT-GTG-AGC-AAA-AGG-CCA-GCA -AAA-GGC-CAG-GAA-CCG-TAA-AAA-3 [26,56] was obtained from Eurogentec (Liege, Belgium). This oligonucleotide is 100% homologous to pUC19 region 2523-2615.…”

“…After that, the recombinase performs a homology search to find a dsDNA having homologous sequence. The global mechanism is reviewed in [12,56,58]. Briefly, the homology recognition and short strand exchange require binding of ATP, allowing filaments to function as rotary motors.…”

“…Biochemical studies with eukaryotic proteins demonstrated that the ssDNA binding protein RPA, if added to the partially formed nucleoprotein filament, promotes the fully assembled Rad51 filament (reviewed in [56,69]). When added before Rad51, RPA binding affinity to ssDNA usually inhibits filament formation of Rad51 and Rad52 is required as a mediator on this RPA-coated ssDNA.…”

Understanding Rad51 function is a prerequisite for progress in cancer research

“…We recently demonstrated that the semi-hydrophobic environment created by ethylene glycol ethers, such as poly-ethylene glycol (PEG), significantly weakens DNA base-stacking strength but not the hydrogen bonding, and also promotes the DNA strand-exchange reaction even in the absence of any recombinase protein (Feng et al, 2019). Interestingly, both RecA and Rad51 elongate DNA by unstacking one base pair of every three, evidencing a weakening of the base-stacking forces (Chen et al, 2008;Xu et al, 2017;Sun et al 2020). Interestingly, we also note that several hydrophobic residues are present in the proximity of the DNA-binding sites of both Rad51 and RecA (the so-called L1 and L2 loops), and, thus, we speculate that these, just like PEG, can provide a hydrophobic environment that may catalyse the strandexchange reaction.…”