Studying the lesion specific bypass of DNA interstrand crosslinks by replicative and translesion synthesis DNA polymerases

Abstract

DNA interstrand crosslinks (ICLs) are covalent linkages between two strands of a DNA duplex. ICLs are induced by chemotherapeutic drugs such as cisplatin, nitrogen mustards or mitomycin C, which induce different types of distortion of into the DNA helix. However, the correlation between degree of helical distortion and choice of DNA polymerase to bypass the ICL lesions has not been studied yet. Dr. Young K. Cheun, a former lab member, investigated which DNA polymerase is required to bypass nitrogen mustard and cisplatin ICL lesions using biochemical experiments. He found that the TLS polymerase POLH can bypass both nitrogen mustard-ICL and cisplatin-ICL. Surprisingly, replicative POLE was able to bypass the nitrogen mustard-ICL lesions, suggesting that TLS polymerases are not needed to bypass all ICLs. POLE was however unable to bypass cisplatin-ICL lesions, demonstrating that polymerase activity on ICLs is structure dependent. We suggest that POLE can bypass nitrogen mustard-ICL because of the flexible structure of this lesion. And we hypothesize that TLS polymerases are not absolutely required to bypass ICLs, and that this requirement depends on the degree of distortion induced into the DNA duplex. Based on this observation, we propose a model for polymerase use in ICL repair. POLE alone can bypass the nitrogen mustard ICL. However, to bypass the cisplatin ICL lesion, pol h and z are required for insertion and extension step, respectively. To test our model in mammalian cells, we generated TLS polymerase KO cell lines using CRISPR-Cas9. We observed that loss of TLS polymerase induced higher cellular sensitivity and increased DNA damage signaling including gH2AX activation upon cisplatin treatment. Loss of REV1 and REV7 increased the sensitivity to nitrogen mustard while POLH deficiency did not. Intriguingly, gH2AX activation by nitrogen mustard treatment was exacerbated in REV1 and REV7 but not in POLH KO cell, following the sensitivity. Additionally, the cellular effect of unresolved ICL lesion in TLS polymerase KO cells will be assessed by detecting global DNA synthesis rate and cell cycle progression.