Abstract LB-323: Control of DNA repair and genome stability by the colon microbiome: The EPEC bacterially encoded NLEE virulence effector protein methylates and inactivates the human ZRANB3 DNA repair helicase

Colorectal cancer develops in the context of probably the most complex microenvironment in the human body: the colon microbiota comprised of >10¹³ bacteria, most of which are uncharacterized. Recently there has been great interest in defining the normal colon microbiome and how it is remodeled during disease states. Both commensal and infective enteropathogenic bacteria utilize a vast array of mechanisms to reduce inflammation in the gut, many of which are directed at the innate immune system, though undoubtedly, other cellular homeostasis systems are targeted. To limit inflammation bacterially encoded virulence proteins are injected into colon epithelial cells: many function by disrupting NFκB signaling. We have characterized the NLEE virulence protein which is encoded by EPEC bacteria and following injection by the T3SS, targets the TAB2 scaffold protein resulting in inactivation of the TAK1 kinase in the NFκB pathway. The Shao laboratory discovered that NLEE is an S-adenosyl-methionine (SAM)-dependent cysteine methyltransferase that methylates a single cysteine in the C4 zinc finger of TAB2 thereby unfolding and abolishing its function. The discovery of this first-in-class, Zinc Finger (ZF)-directed cysteine methytransferase, establishes the paradigm that eukaryotic ZFs can be subject to PTMs at the chelation residues that abolish finger function. We hypothesize that cysteine methylation of ZF domains is widespread and as yet, under appreciated PTM which regulates cellular signal transduction. The crystal structure of NLEE was solved and shows a novel SAM binding motif positioned in a deep cleft which confers a very high specificity for ZF binding and methylation. We searched for novel NLEE ZF-containing substrates in EPEC infected colon cells and discovered that the human DNA repair protein ZRANB3, a helicase which participates in the translesion synthesis repair pathway directly at replication forks, is a robust substrate for NLEE. Methylation of the C4 ZF of ZRANB3 is predicted to abolish its binding to K63-linked ubiquitinated-PCNA at stalled replication forks leading to fork collapse and replication stress. Additional NLEE substrates discovered in this DNA repair pathway include a PCNA-directed OTU class deubiquitinase and the NPL4 segregase complex, both of which limit repair functions. Together with recent work showing that bacterial virulence proteins can also down regulate the DNA mismatch repair system, these results suggest the remarkable conclusion that infectious colon bacteria have the capacity to abolish specific DNA repair systems in the colon epithelium. This work provides a unique and unexpected mechanistic link between the colon microbiome and genome stability in the colon. Whether a remodeled colon cancer specific microbiome contributes to tumor progression via induction of genome instability by inactivating DNA repair pathways is interesting to contemplate. Citation Format: Jayashree Karar, Hongzhuang Peng, Li Zhang, Qing Yao, Ilan Rosenshine, Feng Shao, Frank J. Rauscher. Control of DNA repair and genome stability by the colon microbiome: The EPEC bacterially encoded NLEE virulence effector protein methylates and inactivates the human ZRANB3 DNA repair helicase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-323. doi:10.1158/1538-7445.AM2014-LB-323