DNA and Chromosomes
- Homologous recombination (HR) is a universally conserved DNA repair pathway that can result in the exchange of genetic material. In eukaryotes, HR has evolved into an essential step in meiosis. During meiosis many eukaryotes utilize a two-recombinase pathway. This system consists of Rad51 and the meiosis-specific recombinase Dmc1. Both recombinases have distinct activities during meiotic HR, despite being highly similar in sequence and having closely related biochemical activities, raising the question of how these two proteins can perform separate functions.
- During meiosis, the two DNA recombinases Rad51 and Dmc1 form specialized presynaptic filaments that are adapted for performing recombination between homologous chromosomes. There is currently a limited understanding of how these two recombinases are organized within the meiotic presynaptic filament. Here, we used single molecule imaging to examine the properties of presynaptic complexes composed of both Rad51 and Dmc1. We demonstrate that Rad51 and Dmc1 have an intrinsic ability to self-segregate, even in the absence of any other recombination accessory proteins.
- Rad52 is a highly conserved protein involved in the repair of DNA damage. Human RAD52 has been shown to mediate single-stranded DNA (ssDNA) and is synthetic lethal with mutations in other key recombination proteins. For this study, we used single-molecule imaging and ssDNA curtains to examine the binding interactions of human RAD52 with replication protein A (RPA)-coated ssDNA, and we monitored the fate of RAD52 during assembly of the presynaptic complex. We show that RAD52 binds tightly to the RPA-ssDNA complex and imparts an inhibitory effect on RPA turnover.
- Homologous recombination plays key roles in double-strand break repair, rescue, and repair of stalled replication forks and meiosis. The broadly conserved Rad51/RecA family of recombinases catalyzes the DNA strand invasion reaction that takes place during homologous recombination. We have established single-stranded (ss)DNA curtain assays for measuring individual base triplet steps during the early stages of strand invasion. Here, we examined how base triplet stepping by RecA, Rad51, and Dmc1 is affected by DNA sequence imperfections, such as single and multiple mismatches, abasic sites, and single nucleotide insertions.
- 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.
- Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins.