ATP hydrolysis Promotes Duplex DNA Release by the RecA Presynaptic Complex

Abstract

Homologous recombination is an important DNA repair pathway that plays key roles in maintaining genome stability. Escherichia coli RecA is an ATP-dependent DNA-binding protein that catalyzes the DNA strand exchange reactions in homologous recombination. RecA assembles into long helical filaments on single-stranded DNA, and these presynaptic complexes are responsible for locating and pairing with a homologous duplex DNA. Recent single molecule studies have provided new insights into RecA behavior, but the potential influence of ATP in the reactions remains poorly understood. Here we examine how ATP influences the ability of the RecA presynaptic complex to interact with homologous dsDNA. We demonstrate that over short time regimes, RecA presynaptic complexes sample heterologous dsDNA similarly in the presence of either ATP or ATP␥S, suggesting that initial interactions do not depend on ATP hydrolysis. In addition, RecA stabilizes pairing intermediates in three-base steps, and stepping energetics is seemingly unaltered in the presence of ATP. However, the overall dissociation rate of these paired intermediates with ATP is ϳ4-fold higher than with ATP␥S. These experiments suggest that ATP plays an unanticipated role in promoting the turnover of captured duplex DNA intermediates as RecA attempts to align homologous sequences during the early stages of recombination.Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition (1-4). Homologous recombination contributes to double-strand DNA break repair, the rescue of stalled or collapsed replication forks, programmed and aberrant chromosomal rearrangements, horizontal gene transfer, and meiosis (5-9). The importance of homologous recombination is underscored by its broad conservation across all kingdoms of life and the findings that defects in key recombination proteins can result in a loss of genome integrity and lead to gross chromosomal rearrangements that are a hallmark of cancer in eukaryotes.Many of the key reactions in homologous recombination are catalyzed by the Rad51/RecA family of DNA recombinases (1-4). These recombinases are broadly conserved, and prominent family members include bacterial RecA, the archeal protein RadA, and the eukaryotic recombinases Rad51 and Dmc1. RecA is the archetypal recombinase originally identified in genetic screens for Escherichia coli mutants defective in recombination (10), and much of our current understanding of recombination mechanisms can be attributed to studies of bacterial RecA (4).During recombination, RecA bound to the presynaptic ssDNA must first locate a homologous duplex DNA template. This process is referred to as the homology search, and it is conceptually similar to the target searches of other site-specific DNA-binding proteins (11)(12)(13)(14). Once aligned, the presynaptic ssDNA can be paired with the complementary strand of a homologous dsDNA, resulting in the displacement of the noncomplementary s...

DNA with Different Local Torsional States Affects RecA‐Mediated Recombination Progression

“…Recent single-molecule data also demonstrated the additional function of RecA ATPase activity in homology searching is to stimulate the bypass of heterologous contact by increasing dsDNA turnover,s trand exchange kinetics under ATPi sa ccordingly faster than that in ATPgS. [32] Consistent with these notions, our data showedareductioni ns trand exchange efficiency when ATPh ydrolysis is abolished ( Figure 1B). Previous work using either the 229/149 hybrid DNA or the 427/352h ybrid DNA returnsw ith similar strand exchange efficiencies.…”

“…In the absence of ATP hydrolysis, RecA could not dissociate from DNA and likely can't release torsional strain accumulated in DNA. Recent single‐molecule data also demonstrated the additional function of RecA ATPase activity in homology searching is to stimulate the bypass of heterologous contact by increasing dsDNA turnover, strand exchange kinetics under ATP is accordingly faster than that in ATPγS . Consistent with these notions, our data showed a reduction in strand exchange efficiency when ATP hydrolysis is abolished (Figure B).…”

A change of view: homologous recombination at single-molecule resolution

“…In these studies, D-loop intermediates were generated by incubating RecA, RAD51 or DMC1 presynaptic complexes with fluorescently tagged dsDNA fragments containing an internal microhomology of 8–15 nucleotides in length, which was complementary to the ssDNA. The resulting lifetime changes occurred in 3-nucleotide increments, consistent with the notion that strand exchange intermediates are stabilized by the RAD51/RecA family members in 3-nucleotide steps 92–94 . Moreover, the energetic signature of the stepping behaviour was conserved for RecA, RAD51 and DMC1, indicating that this behaviour appears to be broadly conserved among members of the RAD51/RecA family of DNA recombinases.…”

“…Subsequent analysis of the delay time distributions suggested that strand exchange took place in 3-nucleotide steps 112 . Similar stepping behaviours during strand invasion have been observed using ssDNA curtain assays 92–94 . In these studies, D-loop intermediates were generated by incubating RecA, RAD51 or DMC1 presynaptic complexes with fluorescently tagged dsDNA fragments containing an internal microhomology of 8–15 nucleotides in length, which was complementary to the ssDNA.…”

“…However, a caveat of both approaches is that they do not directly ‘see’ individual strand exchange steps as they are taking place. Rather, the existence of the 3-nucleotide steps is inferred from either a model-dependent analysis of kinetic data 112 or from the stability of the resulting strand exchange products 92–94 . Direct observation of the steps themselves remains a goal of future technical developments.…”