Fig. 1: Mechanisms of stalled fork recovery under conditions of replication stress.

Replication stalling or fork collapse upon exposure to various exogeneous and endogenous stressors activates DNA damage response pathways critical for stalled fork stabilization or restoration and replication restart. a A replication fork encountering an ssDNA break/gap can be recovered by generating a DSB, thereby allowing HR-dependent fork restoration. Nucleases involved in DNA end resection such as MRE11 or EXO1 process the break and generate a 3’ ssDNA overhang necessary to initiate HR at the broken fork. ssDNA-bound RPA is replaced by RAD51 recombinase which in turn allows for strand invasion into the sister chromatid, thereby forming a displacement (D)-loop structure to initiate break-induced replication using the sister chromatid as template. The D-loop can be cleaved by structure-specific endonucleases such as MUS81 to restore the replication fork and continue DNA synthesis. b A dysfunctional fork such as one stalled due to nucleotide depletion is acted upon by the coordinated actions of fork remodeling enzymes and RAD51 to generate a reversed fork intermediate. Fork protecting factors such as BRCA1, BRCA2, RAD51, WRN, and RECQL1 protect reversed forks from unscheduled pathological degradation by nucleases. However, helicase-assisted nucleolytic processing (e.g., WRN-DNA2) of stalled forks (1) in a controlled manner promotes replication restart through HR. Alternatively, a reversed fork can be restored via the branch-migration activity of RECQL1 helicase (2). The reversed fork can also be cleaved by structure-specific endonucleases (e.g., MUS81) to generate a single-ended double strand break (seDSB) at the fork that undergoes homology-directed fork restoration (3), as described in a. Cells genetically deficient in fork-stabilizing proteins (e.g., WRN helicase mutated in WS) accumulate DSBs at forks and experience gross chromosomal instability which may lead to replicative senescence and accelerated aging phenotypes.