In vitro role of Rad54 in Rad51-ssDNA filament-dependent homology search and synaptic complexes formation.

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop).

Dynamic Processing of Displacement Loops during Recombinational DNA Repair.

Displacement loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 h of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps.

Homologous recombination and the repair of DNA double-strand breaks.

Homologous recombination enables the cell to access and copy intact DNA sequence information in , particularly to repair DNA damage affecting both strands of the double helix. Here, we discuss the DNA transactions and enzymatic activities required for this elegantly orchestrated process in the context of the repair of DNA double-strand breaks in somatic cells. This includes homology search, DNA strand invasion, repair DNA synthesis, and restoration of intact chromosomes.

Multi-invasions Are Recombination Byproducts that Induce Chromosomal Rearrangements.

Inaccurate repair of broken chromosomes generates structural variants that can fuel evolution and inflict pathology. We describe a novel rearrangement mechanism in which translocation between intact chromosomes is induced by a lesion on a third chromosome. This multi-invasion-induced rearrangement (MIR) stems from a homologous recombination byproduct, where a broken DNA end simultaneously invades two intact donors.

Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops.

Synthesis-dependent strand annealing (SDSA) is the preferred mode of homologous recombination in somatic cells leading to an obligatory non-crossover outcome, thus avoiding the potential for chromosomal rearrangements and loss of heterozygosity. Genetic analysis identified the Srs2 helicase as a prime candidate to promote SDSA. Here, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion and with a distinct polarity.

Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability.

Never-in-mitosis A-related kinase 1 (Nek1) has established roles in apoptosis and cell cycle regulation. We show that human Nek1 regulates homologous recombination (HR) by phosphorylating Rad54 at Ser572 in late G2 phase. Nek1 deficiency as well as expression of unphosphorylatable Rad54 (Rad54-S572A) cause unresolved Rad51 foci and confer a defect in HR.

RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination.

The Mus81-Mms4 structure-selective endonuclease requires nicked DNA junctions to undergo conformational changes and bend its DNA substrates for cleavage.

Mus81-Mms4/EME1 is a DNA structure-selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81-Mms4 requires inherent rotational flexibility in DNA junctions for optimal catalysis. Förster Resonance Energy Transfer experiments further reveal that recognition of 3'-flap and nicked Holliday junction substrates by Mus81-Mms4 involves induction of a sharp bend with a 100° angle between two duplex DNA arms.

Rad54 functions as a heteroduplex DNA pump modulated by its DNA substrates and Rad51 during D loop formation.

The displacement loop (D loop) is the product of homology search and DNA strand invasion, constituting a central intermediate in homologous recombination (HR). In eukaryotes, the Rad51 DNA strand exchange protein is assisted in D loop formation by the Rad54 motor protein. Curiously, Rad54 also disrupts D loops.

Mus81-Mms4 functions as a single heterodimer to cleave nicked intermediates in recombinational DNA repair.

The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands.

Assays for structure-selective DNA endonucleases.

Structure-selective nucleases perform DNA strand incisions crucial to the repair/resolution of branched DNA molecules arising during DNA replication, recombination, and repair. From a combination of genetics and in vitro nuclease assay studies, we are just beginning to understand how these enzymes recognize their substrates and to identify their in vivo DNA structure targets. By performing nuclease assays on a variety of substrates meant to mimic cellular intermediates, structural features of branched DNA molecules that are important for robust catalysis can be defined.

Conversion of trypsin to a functional threonine protease.

The hydroxyl group of a serine residue at position 195 acts as a nucleophile in the catalytic mechanism of the serine proteases. However, the chemically similar residue, threonine, is rarely used in similar functional context. Our structural modeling suggests that the Ser 195 --> Thr trypsin variant is inactive due to negative steric interaction between the methyl group on the beta-carbon of Thr 195 and the disulfide bridge formed by cysteines 42 and 58.