Structure of the hDmc1-ssDNA filament reveals the principles of its architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination

Crystal structure of Hop2-Mnd1 and mechanistic insights into its role in meiotic recombination

In meiotic DNA recombination, the Hop2-Mnd1 complex promotes Dmc1-mediated single-stranded DNA (ssDNA) invasion into homologous chromosomes to form a synaptic complex by a yet-unclear mechanism. Here, the crystal structure of Hop2-Mnd1 reveals that it forms a curved rod-like structure consisting of three leucine zippers and two kinked junctions. One end of the rod is linked to two juxtaposed winged-helix domains, and the other end is capped by extra ?-helices to form a helical bundle-like structure. Deletion analysis shows that the helical bundle-like structure is sufficient for interacting with the Dmc1-ssDNA nucleofilament, and molecular modeling suggests that the curved rod could be accommodated into the helical groove of the nucleofilament. Remarkably, the winged-helix domains are juxtaposed at fixed relative orientation, and their binding to DNA is likely to perturb the base pairing according to molecular simulations. These findings allow us to propose a model explaining how Hop2-Mnd1 juxtaposes Dmc1-bound ssDNA with distorted recipient double-stranded DNA and thus facilitates strand invasion

Structure and mechanism of human DNA polymerase η

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Pol eta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol eta in replicating through D loop and DNA fragile sites

ruvA Mutants that resolve Holliday junctions but do not reverse replication forks

RuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination

Cooperation of RAD51 and RAD54 in regression of a model replication fork

DNA lesions cause stalling of DNA replication forks, which can be lethal for the cell. Homologous recombination (HR) plays an important role in DNA lesion bypass. It is thought that Rad51, a key protein of HR, contributes to the DNA lesion bypass through its DNA strand invasion activity. Here, using model stalled replication forks we found that RAD51 and RAD54 by acting together can promote DNA lesion bypass in vitro through the ‘template-strand switch’ mechanism. This mechanism involves replication fork regression into a Holliday junction (‘chicken foot structure’), DNA synthesis using the nascent lagging DNA strand as a template and fork restoration. Our results demonstrate that RAD54 can catalyze both regression and restoration of model replication forks through its branch migration activity, but shows strong bias toward fork restoration. We find that RAD51 modulates this reaction; by inhibiting fork restoration and stimulating fork regression it promotes accumulation of the chicken foot structure, which we show is essential for DNA lesion bypass by DNA polymerase in vitro. These results indicate that RAD51 in cooperation with RAD54 may have a new role in DNA lesion bypass that is distinct from DNA strand invasion

Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.

Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. PALB2's tumor-suppressing effect is thought to be based on its ability to facilitate BRCA2's function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to stimulate strand invasion, a vital step of homologous recombination. This stimulation occurs through reinforcing biochemical mechanisms, as PALB2 alleviates inhibition by RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Our studies provide the first biochemical insights into PALB2's function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair

RAD54 controls access to the invading 3′-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae

Rad51 is a key protein in homologous recombination performing homology search and DNA strand invasion. After DNA strand exchange Rad51 protein is stuck on the double-stranded heteroduplex DNA product of DNA strand invasion. This is a problem, because DNA polymerase requires access to the invading 3′-OH end to initiate DNA synthesis. Here we show that, the Saccharomyces cerevisiae dsDNA motor protein Rad54 solves this problem by dissociating yeast Rad51 protein bound to the heteroduplex DNA after DNA strand invasion. The reaction required species-specific interaction between both proteins and the ATPase activity of Rad54 protein. This mechanism rationalizes the in vivo requirement of Rad54 protein for the turnover of Rad51 foci and explains the observed dependence of the transition from homologous pairing to DNA synthesis on Rad54 protein in vegetative and meiotic yeast cells

Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures

Saccharomyces cerevisiae Mph1 is a 3–5′ DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3′ to 5′ directionality, Mph1 displaced 3′-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5′-flap strand more efficiently than the 3′ flap strand from double-flap substrates, which is not expected for a 3–5′ DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5′ single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway

Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology

The defining event in homologous recombination is the exchange of base-paired partners between a single-stranded (ss) DNA and a homologous duplex driven by recombinase proteins, such as human RAD51. To understand the mechanism of this essential genome maintenance event, we analyzed the structure of RAD51–DNA complexes representing strand exchange intermediates at nanometer resolution by scanning force microscopy. Joint molecules were formed between substrates with a defined ssDNA segment and homologous region on a double-stranded (ds) partner. We discovered and quantified several notable architectural features of RAD51 joint molecules. Each end of the RAD51-bound joints had a distinct structure. Using linear substrates, a 10-nt region of mispaired bases blocked extension of joint molecules in all examples observed, whereas 4 nt of heterology only partially blocked joint molecule extension. Joint molecules, including 10 nt of heterology, had paired DNA on either side of the heterologous substitution, indicating that pairing could initiate from the free 3′end of ssDNA or from a region adjacent to the ss–ds junction. RAD51 filaments covering joint ss–dsDNA regions were more stable to disassembly than filaments covering dsDNA. We discuss how distinct structural features of RAD51-bound DNA joints can play important roles as recognition sites for proteins that facilitate and control strand exchange

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase

We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10–13 nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (ΔG° approximately −6.7 kcal/mol). The relative contribution of the oxoG unit of DNA (ΔG° approximately −3.3 kcal/mol) together with other specific interactions (ΔG° approximately −0.7 kcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the kcat term instead; the rate increases by 6–7 orders of magnitude for cognate DNA as compared with non-cognate one. The kcat values for substrates of different sequences correlate with the DNA twist, while the KM values correlate with ΔG° of the DNA fragments surrounding the lesion (position from −6 to +6). The functions for predicting the KM and kcat values for different sequences containing oxoG were found