Molecular cooperation between the Werner syndrome protein and replication protein A in relation to replication fork blockage.
Molecular cooperation between the Werner syndrome protein and replication protein A in relation to replication fork blockage.
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DOI：
10.1074/jbc.m110.105411
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发表时间：
2011-02-04
期刊：
影响因子：
--
通讯作者：
Orren DK
中科院分区：
文献类型：
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作者：
Machwe A;Lozada E;Wold MS;Li GM;Orren DK
早衰和癌症易患的Werner综合征是由RecQ解旋酶家族成员Werner综合征蛋白(WRN)的功能丧失引起的。在细胞水平上，WRN的丢失会导致复制异常和染色体异常，这表明WRN在维持基因组稳定方面发挥了作用。与这一概念一致，WRN在DNA底物上具有退火酶、核酸外切酶和依赖于ATPase的解旋酶活性，对复制和重组结构具有特别高的亲和力和活性。在某些DNA损伤处理之后，WRN被招募到被阻止复制的位置，并与人类单链DNA结合蛋白复制蛋白A(RPA)共定位。在这项研究中，我们研究了WRN和RPA之间的物理和功能相互作用，特别是与复制分叉阻断有关的作用。免疫共沉淀实验表明，阻断DNA复制的损伤性处理大大增加了WRN和RPA在体内的结合，并证实了纯化的WRN和RPA之间的直接相互作用。此外，我们研究了RPA(未修饰和过度磷酸化模拟物)和WRN在模型复制叉和设计用于结合RPA的有间隙的双链底物上的联合作用。即使RPA按化学计量结合到这个缺口上，WRN也有效地催化了叉状底物的退化。进一步的分析表明，WRN可以取代两种底物中的RPA。WRN对RPA的置换与其ATPase和解旋酶依赖的分叉重塑无关。综上所述，我们的结果表明，在复制受阻时，WRN和RPA在功能上相互作用和合作，以帮助适当地解决复制分叉和保持基因组的稳定。
The premature aging and cancer-prone disease Werner syndrome is caused by loss of function of the RecQ helicase family member Werner syndrome protein (WRN). At the cellular level, loss of WRN results in replication abnormalities and chromosomal aberrations, indicating that WRN plays a role in maintenance of genome stability. Consistent with this notion, WRN possesses annealing, exonuclease, and ATPase-dependent helicase activity on DNA substrates, with particularly high affinity for and activity on replication and recombination structures. After certain DNA-damaging treatments, WRN is recruited to sites of blocked replication and co-localizes with the human single-stranded DNA-binding protein replication protein A (RPA). In this study we examined the physical and functional interaction between WRN and RPA specifically in relation to replication fork blockage. Co-immunoprecipitation experiments demonstrated that damaging treatments that block DNA replication substantially increased association between WRN and RPA in vivo, and a direct interaction between purified WRN and RPA was confirmed. Furthermore, we examined the combined action of RPA (unmodified and hyperphosphorylation mimetic) and WRN on model replication fork and gapped duplex substrates designed to bind RPA. Even with RPA bound stoichiometrically to this gap, WRN efficiently catalyzed regression of the fork substrate. Further analysis showed that RPA could be displaced from both substrates by WRN. RPA displacement by WRN was independent of its ATPase- and helicase-dependent remodeling of the fork. Taken together, our results suggest that, upon replication blockage, WRN and RPA functionally interact and cooperate to help properly resolve replication forks and maintain genome stability.