WRN functions in an EXO1-dependent manner to rescue rad50
MMS sensitivity. Physiologically relevant expression levels of WRN helped to rescue the survival phenotypes of the rad50
mutant. Inability of WRN to rescue IR sensitivity indicates that WRN does not operate in the repair of direct strand breaks in the rad50
mutant background. WRN rescue of MMS sensitivity provides new insight to an earlier observation that yEXO1overexpression partially restored MMS resistance of rad50
]. WRN stimulates EXO1 incision of DNA structures associated with stalled or regressed replication forks, leading us to propose that WRN in collaboration with EXO1 is relied upon to prevent deleterious events at DNA replication forks impeded by base alkylation DNA damage.
WRN functional requirements for rescue of rad50
MMS sensitivity are distinct from that for rescue of the dna2-1
mutant in which WRN non-catalytic C-terminal domain was sufficient [7
]. WRN ATPase/helicase activity operating in an EXO1- dependent pathway is responsible for rescue of rad50
to MMS-induced DNA damage, but not IR-induced damage. Thus, catalytic requirements of WRN necessary for a proper response to DNA damage can be genetically separated in the rad50
mutant background. Although biochemical studies with purified proteins and model DNA substrates demonstrated that the interaction of the C-terminal WRN domain with EXO1 is the minimal requirement for stimulation of EXO1 nuclease activity, the genetic requirement for an intact WRN ATPase/helicase domain to rescue MMS sensitivity of the rad50
mutant suggests that the situation in vivo
is likely to be more complex. It is conceivable that the C-terminal domain fragment of WRN is sufficient to stimulate human EXO1 in vitro
, but that the full-length WRN is required for stimulation of yeast EXO1 in vivo
. Possibly, ATPase-dependent translocation to the site of MMS-induced damage or unwinding of blocked replication fork structures is important for WRN to collaborate with EXO1 and help cells cope with replicational stress. In support of a direct role of WRN to deal with replication fork problems, the Monnat lab showed WRN is required for normal replication fork progression after MMS-induced DNA damage or hydroxyurea-induced fork arrest [6
Exo1 and yeast Sgs1 function in alternative pathways for long range 5’ strand resection at a restriction endonuclease-induced DSB [20
]. Cells deficient in the MRX complex are impaired in the initiation of 5’ resection [21
]. It has been shown that 5’ resection in rad50 sgs1
and rad50 exo1
double mutant is even more delayed than in rad50
single mutant, suggesting that in the absence of MRX complex both Sgs1 and Exo1 can still initiate limited DSB end processing. Our results implicate WRN helicase activity operates in the rad50
background in an Exo1-dependent pathway to confer MMS resistance. This is in contrast to Sgs1 in yeast and BLM in human cells that function in parallel with Exo1 to promote DSB resection [19
]. Our studies thus suggest the delineation of WRN and BLM functions in DNA damage repair pathway. Consistent with the inability of helicase/ATPase dead WRN to rescue MMS sensitivity, the helicase domain of Sgs1 is required for resection of DNA ends generated by the trimming function of the MRX/Sae2 at a DSB [20
BLM expression in the rad50
mutant partially restored IR resistance but not MMS resistance, consistent with the possibility that BLM helps cells to cope with strand breaks, but not alkylated base damage induced by MMS. BLM stimulates EXO1 nucleolytic activity on a linearized plasmid DNA molecule [23
]. A delineation of WRN and BLM function is suggested by their differential effects on rad50
MMS and IR sensitivity.
Establishment of a model genetic system for WRN enabled us to investigate the biological consequences of an engineered mutant (K1016A) in the conserved RQC domain as well as a naturally occurring polymorphism (R834C) in the helicase core domain. The R834C polymorphism resulted in a loss of WRN function in DNA damage resistance drawing further attention to this allele as potentially contributing to age-related disease. Thus yeast is a useful model system for studying WRN function in conserved genetic pathways that confer resistance to DNA damaging agents. Moreover, yeast genetics can be used to screen other naturally occurring WRN polymorphisms and engineered WRN domain mutants to decipher WRN functions in genome stability.
Dependence of WRN on EXO1 to rescue rad50
MMS-sensitivity suggested that enhancement of endogenous EXO1 cleavage of DNA structural intermediates associated with stalled replication forks is responsible for genetic complementation. Previously, we showed that WRN interacts with hEXO1 in vivo
and WRN stimulates EXO1 incision of nicked duplex and 5’ flap substrates [17
]. While these substrates represent key intermediates of mismatch repair and Okazaki fragment processing, respectively, we wanted to investigate the effect of WRN on EXO1 processing of DNA structures associated with stalled replication forks. Demonstration that EXO1 is recruited to stalled replication forks in checkpoint defective rad53
] and acts to counteract reversed fork accumulation by generating ssDNA intermediates [48
] suggested that WRN might collaborate with EXO1 to process a stalled replication fork intermediate. On a replication fork structure, WRN stimulated EXO1 5’ incision of what would be the nascent lagging strand arm. In a model for EXO1-mediated processing of stalled forks, EXO1 resects the newly synthesized lagging strand, thereby resolving the sister chromatid junction and preventing reversal at collapsed replication forks [48
An alternative strategy for EXO1 to act with WRN at stalled replication forks is presented when fork regression occurs. Accumulation of chicken foot structures through fork reversal produces abnormal replication intermediates that can be processed by recombination pathways leading to genomic instability in certain mutant backgrounds. WRN stimulated EXO1 5’ incision of the synthetic Holliday Junction, suggesting that the two proteins may collaborate to process chicken foot structures that form as a consequence of fork regression. Altogether, our results unmask a previously unappreciated role of EXO1 in processing stalled and regressed replication forks that form in DNA repair deficient cells. WRN expression in the rad50 background may help to insure appropriate processing of stalled or regressed replication fork structures.
How are these results consistent with characteristics of mutant WRN
cells? WS cells display a reduced rate of repair, elevated apoptotic cell death and increased DNA strand breaks after replication arrest by hydroxyurea treatment or DNA damage induced in S phase [53
]. Expression of a HJ resolvase rescues both the recombination defect and cell survival following DNA damage in WS cells [54
], suggesting that inappropriate processing of stalled replication fork intermediates directly contributes to aberrant homologous recombination characteristic of WS cells. Moreover, suppression of RAD51-dependent recombination significantly improved survival of WS cells following DNA damage [54
]. Replication defects in WS cell lines point to an underlying deficiency when fork progression is abnormal. Co-localization of WRN with arrested replication forks in response to hydroxyurea treatment [55
] or DNA damage [31
] is consistent with its role in response to replicational stress. Asymmetry of DNA replication fork progression in WS cells suggests that WRN acts to prevent collapse of forks or to resolve junctions at stalled forks and that loss of this capacity may be a contributory factor in premature ageing [56
]. Collectively, our findings implicate a role of WRN helicase acting with EXO1 in a conserved pathway to process DNA structures associated with stalled or blocked replication forks.
The genetic results provide new insight to the molecular and cellular roles of WRN helicase in DNA metabolism. In conclusion, WRN helicase function is required in an EXO1-dependent pathway to maintain genomic stability after replicational stress.