Burgers, P. M. | bioRxiv Science

bioRxiv2020

Bypass of DNA Interstrand crosslinks by a Rev1-DNA polymerase ζ complex

DNA polymerase {zeta} (Pol {zeta}) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases {eta}, {zeta}, and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based ICL with an 8-atom linker between the crosslinked bases. The Rev1-Pol {zeta} complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol {zeta} alone or Pol {eta}. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol {eta}, Pol {zeta}, and Rev1-Pol {zeta}. Rev1-Pol {zeta}, and particularly Pol {zeta} alone showed a tendency to stall before the ICL, whereas Pol {eta} stalled just after insertion across the ICL. The stalling of Pol {eta} directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol {eta} providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol {zeta} directly past the ICL was observed, suggesting that the proposed function of Pol {zeta} as an extender DNA polymerase is also required for ICL repair.

biochemistry↗

bioRxiv2019

Modeling Cancer Genomic Data in Yeast Reveals Selection Against ATM Function During Tumorigenesis

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene-induced carcinogenesis. The complex is integral to all modes of double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S.cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was defective while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.\n\nAuthor SummaryA complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair.

genetics↗