Polarity and bypass of DNA heterology during branch migration of Holliday junctions by human RAD54, BLM, and RECQ1 proteins.
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ABSTRACT:
SUBMITTER: Mazina OM
PROVIDER: S-EPMC3320930 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In eukaryotes, RAD54 catalyzes branch migration (BM) of Holliday junctions, a basic process during DNA repair, replication, and recombination. RAD54 also stimulates RAD51 recombinase and has other activities. Here, we investigate the structural determinants for different RAD54 activities. We find that the RAD54 N-terminal domain (NTD) is responsible for initiation of BM through two coupled, but distinct steps; specific binding to Holliday junctions and RAD54 oligomerization. Furthermore, we find that the RAD54 oligomeric state can be controlled by NTD phosphorylation at S49, a CDK2 consensus site, which inhibits RAD54 oligomerization and, consequently, BM. Importantly, the effect of phosphorylation on RAD54 oligomerization is specific for BM, as it does not affect stimulation of RAD51 recombinase by RAD54. Thus, the transition of the oligomeric states provides an important control of the biological functions of RAD54 and, likely, other multifunctional proteins.
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Project description:Genetic recombination occurs between homologous DNA molecules via a four-way (Holliday) junction intermediate. This ancient and ubiquitous process is important for the repair of double-stranded breaks, the restart of stalled replication forks, and the creation of genetic diversity. Once formed, the four-way junction alone can undergo the stepwise exchange of base pairs known as spontaneous branch migration. Conventional ensemble assays, useful for finding average migration rates over long sequences, have been unable to examine the affect of sequence and structure on the migration process. Here, we present a single-molecule spontaneous branch migration assay with single-base pair resolution in a study of individual DNA junctions that can undergo one step of migration. Junctions exhibit markedly different dynamics of exchange between stacking conformers depending on the point of strand exchange, allowing the moment at which branch migration occurs to be detected. The free energy landscape of spontaneous branch migration is found to be highly nonuniform and governed by two types of sequence-dependent barriers, with unmediated local migration being up to 10 times more rapid than the previously deduced average rate.
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Project description:RecQ helicases are essential for the maintenance of genome stability. Five members of the RecQ family have been found in humans, including RECQ1, RECQ5, BLM, WRN, and RECQ4; the last three are associated with human diseases. At this time, only BLM and WRN helicases have been extensively characterized, and the information on the other RecQ helicases has only started to emerge. Our current paper is focused on the biochemical properties of human RECQ1 helicase. Recent cellular studies have shown that RECQ1 may participate in DNA repair and homologous recombination, but the exact mechanisms of how RECQ1 performs its cellular functions remain largely unknown. Whereas RECQ1 possesses poor helicase activity, we found here that the enzyme efficiently promotes DNA branch migration. Further analysis revealed that RECQ1 catalyzes unidirectional three-stranded branch migration with a 3' --> 5' polarity. We show that this RECQ1 activity is instrumental in specific disruption of joint molecules (D-loops) formed by a 5' single-stranded DNA invading strand, which may represent dead end intermediates of homologous recombination in vivo. The newly found enzymatic properties of the RECQ1 helicase may have important implications for the function of RECQ1 in maintenance of genomic stability.
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Project description:The Holliday junction, the key intermediate of recombination, is generated by strand exchange resulting in a covalent connection between two recombining DNA molecules. Translocation of a Holliday junction along DNA, or branch migration, progressively exchanges one DNA strand for another and determines the amount of information that is transferred between two recombining partners. In Escherichia coli, the RuvAB protein complex promotes rapid and unidirectional branch migration of Holliday junctions. We have studied translocation of Holliday junctions using a quantitative biochemical system together with a 'single-molecule' branch migration assay. We demonstrate that RuvAB translocates the junctions through identical DNA sequences in a processive manner with a broad distribution of individual branch migration rates. However, when the complex encounters short heterologous sequences, translocation of the Holliday junctions is impeded. We conclude that translocation of the junctions through a sequence heterology occurs with a probability of bypass being determined both by the length of the heterologous region and the lifetime of the stalled RuvAB complex.
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Project description:The Holliday junction (HJ), or four-way junction, is a central intermediate state of DNA for homologous genetic recombination and other genetic processes such as replication and repair. Branch migration is the process by which the exchange of homologous DNA regions occurs, and it can be spontaneous or driven by proteins. Unfolding of the HJ is required for branch migration. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. Folding of the HJ in one of the folded conformations terminates the branch migration phase. At the same time, in the unfolded state HJ rapidly migrates over entire homology region of the HJ in one hop. This process can be affected by irregularities in the DNA double helical structure, so mismatches almost terminate a spontaneous branch migration. Single-stranded breaks or nicks are the most ubiquitous defects in the DNA helix; however, to date, their effect on the HJ branch migration has not been studied. In addition, although nicked HJs are specific substrates for a number of enzymes involved in DNA recombination and repair, the role of this substrate specificity remains unclear. Our main goal in this work was to study the effect of nicks on the efficiency of HJ branch migration and the dynamics of the HJ. To accomplish this goal, we applied two single-molecule methods: atomic force microscopy and fluorescence resonance energy transfer. The atomic force microscopy data show that the nick does not prevent branch migration, but it does decrease the probability that the HJ will pass the DNA lesion. The single-molecule fluorescence resonance energy transfer approaches were instrumental in detailing the effects of nicks. These studies reveal a dramatic change of the HJ dynamics. The nick changes the structure and conformational dynamics of the junctions, leading to conformations with geometries that are different from those for the intact HJ. On the basis of these data, we propose a model of branch migration in which the propensity of the junction to unfold decreases the lifetimes of folded states, thereby increasing the frequency of junction fluctuations between the folded states.
| S-EPMC2941029 | biostudies-literature
Project description:The Holliday junction is a central intermediate in various genetic processes including homologous, site-specific recombination and DNA replication. Recent single molecule FRET experiments led to the model for branch migration as a stepwise stochastic process in which the branch migration hop is terminated by the folding of the junction. In this article, we studied the effect of the sequence on Holliday junction dynamics and branch migration process. We show that a GC pair placed at the border of the homologous region almost prevents the migration into this position. At the same time, insertion of a GC pair into the middle of the AT tract does not show this effect, however when the junction folds at this position, it resides at this position much longer time in comparison to the folding at AT pairs. Two contiguous GC pairs do not block migration as well and generally manifest the same effect as one GC pair--the junction when it folds resides at these positions for a relatively long time. The same elevated residence time was obtained for the design with the homology region that consists of only GC pairs. These data suggest a model for branch migration in which the sequence modulates the overall stochastic process of the junction dynamics and branch migration by the variability of the time that the junction dwells before making a migration hop.
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Project description:Cancer cells can activate the alternative lengthening of telomeres (ALT) pathway to promote replicative immortality. The ALT pathway promotes telomere elongation through a homologous recombination pathway known as break-induced replication (BIR), which is often engaged to repair single-ended double-stranded breaks (DSBs). Single-ended DSBs are resected to promote strand invasion and facilitate the formation of a local displacement loop (D-loop), which can trigger DNA synthesis, and ultimately promote telomere elongation. However, the exact proteins involved in the maturation, migration, and resolution of D-loops at ALT telomeres are unclear. In vitro, the DNA translocase RAD54 both binds D-loops and promotes branch migration suggesting that RAD54 may function to promote ALT activity. Here, we demonstrate that RAD54 is enriched at ALT telomeres and promotes telomeric DNA synthesis through its ATPase-dependent branch migration activity. Loss of RAD54 leads to the formation of unresolved recombination intermediates at telomeres that form ultra-fine anaphase bridges in mitosis. These data demonstrate an important role for RAD54 in promoting ALT-mediated telomere synthesis.
| S-EPMC7271314 | biostudies-literature
Project description:The Holliday junction (HJ) is a central intermediate in various genetic processes including homologous and site-specific recombination and DNA replication. Branch migration allows the exchange between homologous DNA regions, but the detailed mechanism for this key step of DNA recombination is unidentified. Here, we report direct real-time detection of branch migration in individual molecules. Using appropriately designed HJ constructs we were able to follow junction branch migration at the single-molecule level. Branch migration is detected as a stepwise random process with the overall kinetics dependent on Mg2+ concentration. We developed a theoretical approach to analyze the mechanism of HJ branch migration. The data show steps in which the junction flips between conformations favorable to branch migration and conformations unfavorable to it. In the favorable conformation (the extended HJ geometry), the branch can migrate over several base pairs detected, usually as a single large step. Mg2+ cations stabilize folded conformations and stall branch migration for a period considerably longer than the hopping step. The conformational flip and the variable base pair hopping step provide insights into the regulatory mechanism of genetic processes involving HJs.
| S-EPMC1140338 | biostudies-literature
Project description:The Rad51 and Rad54 proteins play important roles during homologous recombination in eukaryotes. Rad51 forms a nucleoprotein filament on single-stranded DNA and performs the initial steps of double strand break repair. Rad54 belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that Rad54 promotes branch migration of Holliday junctions. Here we find that human Rad51 (hRad51) significantly stimulates the branch migration activity of hRad54. The stimulation appears to be evolutionarily conserved, as yeast Rad51 also stimulates the branch migration activity of yeast Rad54. We further investigated the mechanism of this stimulation. Our results demonstrate that the stimulation of hRad54-promoted branch migration by hRad51 is driven by specific protein-protein interactions, and the active form of the hRad51 filament is more stimulatory than the inactive one. The current results support the hypothesis that the hRad51 conformation state has a strong effect on interaction with hRad54 and ultimately on the function of hRad54 in homologous recombination.
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Project description:The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.
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