The Role of Human RAD52 Protein in Genome Stability

University Of Iowa, Iowa City IA

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Abstract

ABSTRACT The accurate and timely DNA replication program is a prerequisite of a stable genome. This research project is built around our discovery that the human DNA repair protein RAD52 performs an important and previously unknown function in supporting DNA replication. RAD52 promotes genome integrity through several biochemical and cellular functions. As a gatekeeper of replication forks, RAD52 binds to and stabilizes stalled or damaged replication forks protecting them from reversal by SMARCAL1, and subsequent MRE11-dependent degradation. RAD52 absence promotes RAD51-dependent recruitment and activation of DNA polymerase alpha/primase and an alternative replication restart pathway that leaves behind large ssDNA gaps. Finally, upon prolonged replication stress, RAD52 cooperates with MUS81 nuclease to cleave the fork producing DNA breaks that can be repaired by homologous recombination. Depletion or pharmacological inhibition of RAD52 results in fork restoration with large ssDNA gaps, genome instability, and selective toxicity in cancer cells displaying BRCAness phenotype or defects in ATM (ataxia–telangiectasia mutated) serine/threonine kinase. We discovered that the replication fork structure enables a unique head-to-head arrangement of two undecameric RAD52 rings. This organization juxtaposes the RAD52 DNA binding sites creating an extended positively charged surface. In addition to DNA binding, this surface is involved in the interaction with MUS81 nuclease. We propose that the two-ring RAD52 structure plays two distinct functions: (1) it enables a dynamic DNA strand exchange reaction which locally remodels the stalled fork, and (2) it positions MUS81 for cleavage. The transition between a spool-like two-ring arrangement to a single ring is a switch between fork protection and mutagenic single-strand annealing activities of RAD52. Our goal for the next funding period is to develop a comprehensive mechanistic understanding of the RAD52 function at the replication fork. In Aim 1 we will combine cryo-electron microscopy (CryoEM), single-molecule biophysics, super-resolution microscopy, biochemical and cell-based analyses with computational modeling to build the structure-activity relationship (SAR) of the nucleoprotein complexes containing fork DNA, RAD52 and ssDNA binding protein RPA. In Aim 2 we will dissect functionally of the MUS81-RAD52 axes. We will map the MUS81 cleavage sites on stalled replication forks protected by RAD52 and RPA, will describe the architecture of the RAD52/fork/MUS81 complexes, and will investigate the MUS81-RAD52 interactions in cells using super- resolution microscopy and proximity labelling. In Aim 3 we will test the hypothesis that RAD52 phosphorylation by c-ABL tyrosine kinase at Y104 prevents formation of the two-ring RAD52-fork structure and switches the RAD52 function from fork protection to single-strand annealing. By completing the proposed studies, we will learn how RAD52 functions at replication forks, how it contributes to genome stability, and will obtain SAR crucial for development of novel, function-specific inhibitors.