Highlights d DNA polymerase q is essential for mutagenic replication through UV lesions d UV-induced skin cancer formation arises in the absence of Polq d Translesion synthesis by Polh or Polq prevents replication fork collapse d TLS protects against chromosomal instability and tumorigenesis

Background: Little is known of biological functions of DNA polymerase (pol) in human cells. Results: pol promotes replication through the common oxidation product, thymine glycol, in human cells. Conclusion: pol active site can accommodate the considerable distortion imposed by thymine glycol at the extension step. Significance: pol may function in translesion synthesis opposite a variety of DNA lesions.

N3-Methyladenine (3-MeA) is formed in DNA by reaction with -adenosylmethionine, the reactive methyl donor, and by reaction with alkylating agents. 3-MeA protrudes into the DNA minor groove and strongly blocks synthesis by replicative DNA polymerases (Pols). However, the mechanisms for replicating through this lesion in human cells remain unidentified. Here we analyzed the roles of translesion synthesis (TLS) Pols in the replication of 3-MeA-damaged DNA in human cells. Because 3-MeA has a short half-life, we used the stable 3-deaza analog, 3-deaza-3-methyladenine (3-dMeA), which blocks the DNA minor groove similarly to 3-MeA. We found that replication through the 3-dMeA adduct is mediated via three different pathways, dependent upon Polι/Polκ, Polθ, and Polζ. As inferred from biochemical studies, in the Polι/Polκ pathway, Polι inserts a nucleotide (nt) opposite 3-dMeA and Polκ extends synthesis from the inserted nt. In the Polθ pathway, Polθ carries out both the insertion and extension steps of TLS opposite 3-dMeA, and in the Polζ pathway, Polζ extends synthesis following nt insertion by an as yet unidentified Pol. Steady-state kinetic analyses indicated that Polι and Polθ insert the correct nt T opposite 3-dMeA with a much reduced catalytic efficiency and that both Pols exhibit a high propensity for inserting a wrong nt opposite this adduct. However, despite their low fidelity of synthesis opposite 3-dMeA, TLS opposite this lesion replicates DNA in a highly error-free manner in human cells. We discuss the implications of these observations for TLS mechanisms in human cells.

Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans.

We demonstrated that the CCK2i4svR is a potential target for PRRT using a radiolabelled sulfated CCK8 peptide. As this receptor is expressed on colorectal and pancreatic tumours, but not in normal tissue, these tumours are potentially new targets for PRRT with CCK8 and gastrin analogs.

Previously, we have shown that human DNA polymerase (Pol) η has two functional PCNA binding motifs, PIP1 and PIP2, and that a C-terminal deletion of Polη that lacks the ubiquitin-binding UBZ domain and the PIP2 domain but retains the PIP1 domain promotes normal levels of translesion synthesis (TLS) opposite a cis-syn TT dimer in human cells. Here, we identify two PIP domains in Polκ, and show that TLS occurs normally in human fibroblast cells in which the pip1 or pip2 mutant Polκ is expressed, but mutational inactivation of both PIP domains renders Polκ non-functional in TLS opposite the thymine glycol lesion. Thus, the two PIP domains of Polκ function redundantly in TLS opposite this DNA lesion in human cells. However, and surprisingly, whereas mutational inactivation of the PIP1 domain completely inhibits the stimulation of DNA synthesis by Polκ in the presence of PCNA, RFC, and RPA, mutations in PIP2 have no adverse effect on PCNA-dependent DNA synthesis. This raises the possibility that activation of Polκ PIP2 as a PCNA binding domain occurs during TLS in human cells, and that protein-protein interactions and post transcriptional modifications are involved in such activation.

Acrolein, an , unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is an ubiquitous environmental pollutant. The reaction of acrolein with the N 2 of guanine in DNA leads to the formation of -hydroxy-1-N 2 -propano-2' deoxyguanosine (-HOPdG) which can exist in DNA in a ring-closed or a ring-opened form. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) which conduct replication through the permanently ringopened reduced form of -HOPdG [(r) -HOPdG] and show that replication through this adduct is mediated via Rev1/Pol, Pol/Pol, and Pol dependent pathways, respectively. Based upon biochemical and structural studies, we propose a role for Rev1 and Pol in inserting a nucleotide (nt) opposite the adduct and for Pols  and  in extending synthesis from the inserted nt in the respective TLS pathway. Based upon genetic analyses and biochemical studies with Pol, we infer a role for Pol at both the nt insertion and extension steps of TLS. Whereas purified Rev1 and Pol primarily incorporate a C opposite (r) -HOPdG, Pol incorporates a C or a T opposite the adduct; nevertheless, TLS mediated by the Pol dependent pathway as well as by other pathways occurs in a predominantly error-free manner in human cells. We discuss the implications of these observations for the mechanisms that could affect the efficiency and fidelity of TLS Pols.Acrolein, an ,-unsaturated aldehyde, is a ubiquitous environmental pollutant formed by incomplete combustion of organic materials and it occurs in the environment as a component of tobacco smoke and automobile exhaust. Moreover, acrolein is generated endogenously as the end product of lipid peroxidation and during the metabolic oxidation of polyamines (1-5). Acrolein adducts have been detected in DNA from a variety of tissues in rats, mice, and humans, indicating that this DNA adduct is generated in vivo from cellular reactions (1-3,6,7).The reaction of acrolein with the N 2 of guanine in DNA followed by ring closure results in the formation of the cyclic adduct -hydroxy-1, N 2 -propano-2'-deoxyguanosine (-HOPdG). -HOPdG can exist in DNA in a ring-closed or a http://www.jbc.org/cgi/doi/10.1074/jbc.RA117.000962 The latest version is at JBC Papers in Press. Published on January 12, 2018 as Manuscript RA117.000962Copyright 2018 by The American Society for Biochemistry and Molecular Biology, Inc.by guest on May 11, 2018 http://www.jbc.org/ Downloaded from 2 ring-opened form (8-10). -HOPdG presents a strong block to synthesis by replicative DNA polymerases and it is also inhibitory to synthesis by yeast and human Pol, particularly at the nucleotide (nt) incorporation step (11,12). DNA synthesis opposite -HOPdG, however, can be mediated by the sequential action of Pols  and , in which Pol incorporates a nt opposite -HOPdG and Pol performs the subsequent extension step (12). In the presence of a reducing agent, -HOPdG can be trapped as the N 2 -(3-hydroxy prop...

Expression of gastrin and cholecystokinin 2 (CCK 2 ) receptor splice variants (CCK 2 R and CCK 2i4sv R) are upregulated in human colonic adenomas where they are thought to contribute to tumor growth and progression. To determine the effects of ectopic CCK 2 receptor variant expression on colonic epithelial cell growth in vitro and in vivo, we employed the non-tumorigenic colonic epithelial cell line, NCM356. Receptor expression was induced using a retroviral expression vector containing cDNAs for either CCK 2i4sv R or CCK 2 R. The adenoma-to carcinoma-multistage sequence of colorectal cancer development is characterized by specific histopathologic criteria as well as defined genetic mutations, which result in the activation of oncogenes (e.g., K-ras) and inactivation of tumor suppressors [e.g., adenomatous polyposis coli (APC) and p53]. 1 It is now well recognized that the key initiating events that underlie most cases of colorectal tumorigenesis, whether familial or sporadic, are the mutations within the Wnt/APC/b-catenin signaling pathway.2 However, increasing evidence also indicate that epigenetic changes in DNA and/or chromatin structure, causing aberrant mRNA splicing and/or inappropriate expression of normal genes, can interact with genetic mutations to contribute to the development of the malignant phenotype.3 Aberrant expression of the gastrin/cholecystokinin 2 (CCK 2 ) receptor, gastrin and its biosynthetic precursors, in a majority of pre-malignant adenomatous polyps strongly implicate a relevant role for this signaling axis in the adenoma-carcinoma sequence. [4][5][6] Carboxyl-terminus-amidated gastrin (i.e., mature gastrin) is produced from the cleavage and post-translational processing of a preprohormone protein. Although both gastrin precursors and mature gastrin are prevalent during the early stages of malignant transformation, as well as in established