Carol A. Parsons

Precise binding of single‐stranded DNA termini by human RAD52 protein

The human RAD52 protein, which exhibits a heptameric ring structure, has been shown to bind resected double strand breaks (DSBs), consistent with an early role in meiotic recombination and DSB repair. In this work, we show that RAD52 binds single‐stranded and tailed duplex DNA molecules via precise interactions with the terminal base. When probed with hydroxyl radicals, ssDNA–RAD52 complexes exhibit a four‐nucleotide repeat hypersensitivity pattern. This unique pattern is due to the interaction of RAD52 with either a 5′ or a 3′ terminus of the ssDNA, is sequence independent and is phased precisely from the terminal nucleotide. Hypersensitivity is observed over ∼36 nucleotides, consistent with the length of DNA that is protected by RAD52 in nuclease protection assays. We propose that RAD52 binds DNA breaks via specific interactions with the terminal base, leading to the formation of a precisely organized ssDNA–RAD52 complex in which the DNA lies on an exposed surface of the protein. This protein–DNA arrangement may facilitate the DNA–DNA interactions necessary for RAD52‐mediated annealing of complementary DNA strands.

The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single‐stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single‐strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.

Resolution of model holliday junctions by yeast endonuclease is dependent upon homologous DNA sequences

Holliday junctions, in which two double-stranded DNA molecules are linked by single-stranded crossovers, are thought to be central intermediates in genetic recombination. We report here the in vitro specificity of a yeast endonuclease for structures analogous to Holliday junctions. Plasmids that extrude inverted repeat sequences into cruciform junctions are cleaved by the introduction of nicks into strands of like polarity, approximately 4-8 nucleotides from the base of the junction. In all cases, cleavage occurs within homologous sequences, and with precise symmetry across the junction. In contrast, a junction containing four arms of unrelated sequence is cleaved asymmetrically. The dependence upon homology for symmetrical cleavage is not found with T4 endonuclease VII, which cleaves branched structures in vitro. Holliday junction resolution appears to occur in a concerted manner by the introduction of nicks into two homologous DNA helices held in alignment.

Resolution of model Holliday junctions by yeast endonuclease: effect of DNA structure and sequence.

The resolution of Holliday junctions in DNA involves specific cleavage at or close to the site of the junction. A nuclease from Saccharomyces cerevisiae cleaves model Holliday junctions in vitro by the introduction of nicks in regions of duplex DNA adjacent to the crossover point. In previous studies [Parsons and West (1988) Cell, 52, 621‐629] it was shown that cleavage occurred within homologous arm sequences with precise symmetry across the junction. In contrast, junctions with heterologous arm sequences were cleaved asymmetrically. In this work, we have studied the effect of sequence changes and base modification upon the site of cleavage. It is shown that the specificity of cleavage is unchanged providing that perfect homology is maintained between opposing arm sequences. However, in the absence of homology, cleavage depends upon sequence context and is affected by minor changes such as base modification. These data support the proposed mechanism for cleavage of a Holliday junction, which requires homologous alignment of arm sequences in an enzyme–DNA complex as a prerequisite for symmetrical cleavage by the yeast endonuclease.

Resolution of Holliday Junctions by the E. coli RuvC Protein

During genetic recombination, intermediates are formed in which two recombining DNA molecules are linked by a Holliday junction (Holliday 1964). There are two general questions regarding the mechanism by which Holliday junctions are resolved to form recombinant products: first, how is the junction recognized, and second, what are the biochemical mechanisms of the cleavage and ligation reactions. The recent identification of a Holliday junction-specific endonuclease from E. coli allows a detailed investigation of these problems and provides new insight into the mechanics of the late steps of genetic recombination.

Proteins from Yeast and Human Cells Specific for Model Holliday Junctions in DNA

Genetic recombination involves the exchange of genetic material between chromosomes to produce new assortments of alleles. As such, it affects one of the most fundamental and important components of heredity, the genome itself. Genetic rearrangements can be favourable or unfavourable, and certain forms of cancer have been linked to gene translocations. To understand the molecular basis of recombination, we have directed our efforts to try to determine how simple organisms recombine their DNA. In bacteria and lower eukaryotes, the enzymes involved in genetic recombination also play a role in the repair of DNA following irradiation or chemical damage. This overlap between recombination and repair is indicative of a need for recombinational repair, a process which ensures that the integrity of the chromosomal material is maintained.