RAD51 Protein ATP Cap Regulates Nucleoprotein Filament Stability

RAD51 mediates homologous recombination by forming an active DNA nucleoprotein filament (NPF). A conserved aspartate that forms a salt bridge with the ATP γ-phosphate is found at the nucleotide-binding interface between RAD51 subunits of the NPF known as the ATP cap. The salt bridge accounts for the nonphysiological cation(s) required to fully activate the RAD51 NPF. In contrast, RecA homologs and most RAD51 paralogs contain a conserved lysine at the analogous structural position. We demonstrate that substitution of human RAD51(Asp-316) with lysine (HsRAD51(D316K)) decreases NPF turnover and facilitates considerably improved recombinase functions. Structural analysis shows that archaebacterial Methanococcus voltae RadA(D302K) (MvRAD51(D302K)) and HsRAD51(D316K) form extended active NPFs without salt. These studies suggest that the HsRAD51(Asp-316) salt bridge may function as a conformational sensor that enhances turnover at the expense of recombinase activity

TRAIP is a master regulator of DNA interstrand crosslink repair

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2–7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles

TRAIP is a master regulator of DNA interstrand crosslink repair

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2–7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles

Subunit Interface Residues F129 and H294 of Human RAD51 Are Essential for Recombinase Function

RAD51 mediated homologous recombinational repair (HRR) of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. RAD51 forms a nucleoprotein filament (NPF) that catalyzes the fundamental homologous pairing and strand exchange reaction (recombinase) required for HRR. Based on structural and functional homology with archaeal and yeast RAD51, we have identified the human RAD51 (HsRAD51) subunit interface residues HsRad51(F129) in the Walker A box and HsRad51(H294) in the L2 ssDNA binding region as potentially important participants in salt-induced conformational transitions essential for recombinase activity. We demonstrate that the HsRad51(F129V) and HsRad51(H294V) substitution mutations reduce DNA dependent ATPase activity and are largely defective in the formation of a functional NPF, which ultimately eliminates recombinase catalytic functions. Our data are consistent with the conclusion that the HsRAD51(F129

Cation-induced conformational rearragement of conserved amino acid residues of RadA.

<p>(<b>A</b>) Subunit interface of MvRadA (MvRAD51) structure in the absence of potassium cation (PDB code 1T4G). (<b>B</b>) Subunit interface region of MvRAD51 structure in the presence of potassium cation (PDB code 1XU4). Structural figures were generated using Pymol. (<b>C</b>) Sequence alignment of WalkerA/P-loop and L2 ssDNA binding region of <i>H. sapiens</i> (Hs), <i>S. cerevisiae</i> (Sc) and <i>M. voltae</i> (Mv) recombinases. HsRAD51 residues F129 and H294 are indicated with asterisks.</p

F129 and H294 of HsRAD51 are critical for DNA binding in the presence of ATP.

<p>(<b>A</b>) ssDNA binding analysis of <i>wild type</i> and HsRAD51 substitution mutant proteins by surface Plasmon resonance (SPR, Biacore) in the absence of an adenine nucleotide, in the presence of ADP and in the presence of ATP. Association and dissociation curve corresponding to 800 nM of each protein is shown. (<b>B</b>) dsDNA binding analysis of <i>wild type</i> and HsRAD51 substitution mutant proteins.</p

Summary of ATP hydrolysis and nucleotide binding data of HsRAD51 <i>wild type</i> and HsRAD51(F129V) and HsRAD51(H294V) mutant proteins.

<p>Summary of ATP hydrolysis and nucleotide binding data of HsRAD51 <i>wild type</i> and HsRAD51(F129V) and HsRAD51(H294V) mutant proteins.</p

Summary of DNA binding data of HsRAD51 <i>wild type</i> and HsRAD51(F129V) and HsRAD51(H294V) mutant proteins.^*

*<p>In some cases, binding and/or dissociation occurs very rapidly, and cannot be accurately determined by SPR. For these cases we simply report a lower bound for <i>k_on</i> and <i>k_off</i>, and cannot determine an accurate dissociation constant, <i>K_D</i> (Indicated by NA).</p

HsRAD51(F129V) and HsRAD51(H294V) are deficient in D-loop formation and strand exchange.

<p>(<b>A</b>) <i>In vitro</i> D-loop assay reaction schematic. (<b>B</b>) 0.8 µM of HsRAD51, HsRAD51(F129V), or HsRAD51(H294V) and [P³²]-labeled ssDNA (90mer; 2.4 µM nt) were preincubated for 10 min at 37°C in the reaction buffer containing 1 mM ATP and 1 mM MgCl₂ or CaCl₂. Reactions were initiated by the addition of supercoiled pBS SK(-) plasmid DNA (35 µM bp). After 15 min, reactions were terminated by the addition of proteinase-K and SDS. Joint molecules (JMs) were analyzed on a 0.9% agarose gel. (<b>C</b>) Analysis of salt and RPA requirement for strand exchange. Reaction schematic shown above: HsRAD51 (5 µM) and φX174 circular ssDNA (30 µM nt) were pre-incubated with 2.5 mM ATP and 1 mM MgCl₂ at 37°C for 5 m prior to the addition of 150 mM NaNH₄HPO₄ (if indicated) and linear φX174 dsDNA (15 µM bp). After 5 m, HsRPA (2 µM) was added (if indicated) and the incubation was continued for 3 h. Samples were deproteinized and analyzed on 0.9% agarose gel with 0.1 µg/mL ethidium bromide.</p

Mutation of HsRAD51(F129) and HsRAD51(H294) residues affect ATP turnover.

<p>(<b>A</b>) Purification of <i>wild type</i> and HsRAD51 substitution mutant proteins. Protein (1 µg) analyzed by 12% SDS-PAGE. (<b>B</b>) Steady-state ATPase activity with ssDNA in the presence of 150 mM KCl. (<b>C</b>) ATPγS binding by <i>wild type</i> and HsRAD51 substitution mutant proteins in the presence of ssDNA and 150 mM KCl. (<b>D</b>) ADP binding by <i>wild type</i> and HsRAD51 substitution mutant proteins in the presence of ssDNA and 150 mM KCl. (<b>E</b>) ATP turnover (<i>k_cat</i>) with ssDNA and dsDNA in the presence and absence of KCl (K⁺). <i>k_cat</i> values were calculated by Michaelis-Menten analysis. Error bars indicate standard deviation from at least three independent experiments.</p