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.
Highlights
Replication stress causes replication fork stalling, resulting in an accumulation of singlestranded DNA
Our previous study has revealed that CST localizes at stalled forks and that the DNA-binding activity of CST is needed for recruitment of RAD51 to stalled forks in response to hydroxyurea (HU) treatment[31]
Our data indicate that CST can recruit RAD51 to Replication protein A (RPA)-bound singlestranded DNA (ssDNA), it lacks recombination mediator activity. Both RPA and CST harbor multiple OB-fold domains and form a heterotrimeric complex. Both complexes exhibit a high affinity for ssDNA, the CST complex presents a differential response to ssDNA depending on the length and sequence of this latter
Summary
Replication stress causes replication fork stalling, resulting in an accumulation of singlestranded DNA (ssDNA). Our findings provide mechanistic insights into how CST targets and tethers RAD51 to RPA-coated ssDNA in response to replication stress. The stalled forks undergo a reversal process that is regulated by RPA, RAD51 and DNA translocases to form a four-way junction structure[5,6,7,8,9,10]. RAD51, a conserved general recombinase, is a central player in promoting fork reversal and in protecting reversed forks from nucleolytic attack[16,17,18,19,20], and it is responsible for DNA doublestrand break (DSB) repair via homologous recombination (HR). Association of RAD51 with stressed forks is necessary for fork reversal and for protecting reversed forks from
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