Crosstalk between CST and RPA regulates RAD51 activity during replication stress

Nat Commun. 2021 Nov 5;12(1):6412. doi: 10.1038/s41467-021-26624-x.

Abstract

Replication stress causes replication fork stalling, resulting in an accumulation of single-stranded DNA (ssDNA). Replication protein A (RPA) and CTC1-STN1-TEN1 (CST) complex bind ssDNA and are found at stalled forks, where they regulate RAD51 recruitment and foci formation in vivo. Here, we investigate crosstalk between RPA, CST, and RAD51. We show that CST and RPA localize in close proximity in cells. Although CST stably binds to ssDNA with a high affinity at low ionic strength, the interaction becomes more dynamic and enables facilitated dissociation at high ionic strength. CST can coexist with RPA on the same ssDNA and target RAD51 to RPA-coated ssDNA. Notably, whereas RPA-coated ssDNA inhibits RAD51 activity, RAD51 can assemble a functional filament and exhibit strand-exchange activity on CST-coated ssDNA at high ionic strength. Our findings provide mechanistic insights into how CST targets and tethers RAD51 to RPA-coated ssDNA in response to replication stress.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Replication / genetics
  • DNA Replication / physiology
  • Electrophoretic Mobility Shift Assay
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • Protein Binding
  • Rad51 Recombinase / genetics
  • Rad51 Recombinase / metabolism*
  • Replication Protein A / genetics
  • Replication Protein A / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • Replication Protein A
  • Saccharomyces cerevisiae Proteins
  • Rad51 Recombinase