Biology subjects

Kowalczykowski, S. C.

Publications and source records attributed to Kowalczykowski, S. C..

3 recordsLinked to original sources

bioRxiv2022

BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA

Mutations in the breast cancer susceptibility gene, BRCA2, greatly increase an individuals lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, Replication Protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins--of which BRCA2 is the defining member in humans --alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to--and the assembly of RAD51 filaments on--a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short pre-assembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation. SignificanceDespite decades of genetic and cell biological studies, mechanistic biochemical analyses of human BRCA2 function in recombinational DNA repair have only been possible since the purification of full-length BRCA2. These mechanistic studies crucially inform with respect to the molecular function of BRCA2 in genome maintenance. Here, we use single-molecule methods to visualize the assembly of RAD51 on individual RPA-coated ssDNA molecules and to see how this process is regulated by the tumor suppressor protein, BRCA2. We show that BRCA2 serves as a chaperone to nucleate RAD51 and deliver it to RPA-coated ssDNA. This work advances understanding of the molecular functions of BRCA2 and, consequently, the molecular etiology of breast cancer in an important way.

biochemistry↗

bioRxiv2022

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

RecA-mediated Homologous Recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report that neither of the two paralogous pneumococcal SSBs could assist SpRecA polymerization on ssDNA. Instead, we found that the conserved RadA helicase promotes this SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATP{gamma}S. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.

microbiology↗

bioRxiv2022

Direct Visualization of Translesion DNA Synthesis Polymerase IV at the Replisome

In bacterial cells, DNA damage tolerance is manifested by the action of translesion DNA polymerases that can synthesize DNA across template lesions that typically block the replicative DNA polymerase III. It has been suggested that one of these TLS DNA polymerases, DNA polymerase IV, can either act in concert with the replisome, switching places on the {beta} sliding clamp with DNA polymerase III to bypass the template damage, or act subsequent to the replisome skipping over the template lesion in the gap in nascent DNA left behind as the replisome continues downstream. Evidence exists in support of both mechanisms. Using single-molecule analyses we show that DNA polymerase IV associates with the replisome in a concentration-dependent manner and remains associated over long stretches of replication fork progression under unstressed conditions. This association slows the replisome, requires DNA polymerase IV binding to the {beta} clamp but not its catalytic activity, and is reinforced by the presence of the {gamma} subunit of the {beta} clamp-loading DnaX complex in the DNA polymerase III holoenzyme. Thus, DNA damage is not required for association of DNA polymerase IV with the replisome. We suggest that under stress conditions such as induction of the SOS response, the association of DNA polymerase IV with the replisome provides both a surveillance/bypass mechanism and a means to slow replication fork progression, thereby reducing the frequency of collisions with template damage and the overall mutagenic potential. SignificanceDamage to the nucleotide bases that make up the DNA in chromosomes creates a problem for their subsequent accurate duplication each time a cell divides. Typically, the cellular enzymatic machinery that replicates the DNA cannot copy a damaged base and specialized trans-lesion DNA polymerases, which are prone to making errors that result in mutations, are required to copy the damaged base, allowing replication to proceed. We demonstrate that the bacterial replisome, which is comprised of the enzymes required to replicate the chromosome, can associate with one of these specialized trans-lesion polymerases over long distances of replicated DNA. This association slows the speed of replication, thereby reducing the chance of mutations arising in the cell under conditions of stress.

biochemistry↗