polι, a remarkably error-prone human DNA polymerase

Abstract

The Saccharomyces cerevisiae RAD30 gene encodes DNA polymerase η. Humans possess two Rad30 homologs. One (RAD30A/POLH) has previously been characterized and shown to be defective in humans with the Xeroderma pigmentosum variant phenotype. Here, we report experiments demonstrating that the second human homolog (RAD30B), also encodes a novel DNA polymerase that we designate polι. polι, is a distributive enzyme that is highly error-prone when replicating undamaged DNA. At template G or C, the average error frequency was ∼1 × 10−2. Our studies revealed, however, a striking asymmetry in misincorporation frequency at template A and T. For example, template A was replicated with the greatest accuracy, with misincorporation of G, A, or C occurring with a frequency of ∼1 × 10−4 to 2 × 10−4. In dramatic contrast, most errors occurred at template T, where the misincorporation of G was, in fact, favored ∼3:1 over the correct nucleotide, A, and misincorporation of T occurred at a frequency of ∼6.7 × 10−1. These findings demonstrate that polι is one of the most error-prone eukaryotic polymerases reported to date and exhibits an unusual misincorporation spectrum in vitro.

Structural Approach To Identify a Lead Scaffold That Targets the Translesion Synthesis Polymerase Rev1

“…In humans, multiple TLS DNA polymerases have been identified that belong to A, B, X, and Y families . The Y-family polymerases Rev1, Polη, Polι, and Polκ and the B-family polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) are responsible for the replicative bypass of the majority of DNA lesions in the process of Rev1/Polζ-dependent TLS. , Rev1 is a Y-family DNA polymerase with limited deoxycytidyltransferase activity whose primary function is to serve as a scaffold for assembly of the multiprotein TLS complex and regulate recruitment of other TLS polymerases through multiple protein–protein interactions (PPIs). , Most of these essential PPIs with other TLS DNA polymerases are mediated through the C-terminal domain of Rev1 (Rev1-CT). This versatile domain can bind Rev1-interacting regions (RIRs) from the Y-family polymerases Polη, Polι, and Polκ and the PolD3 subunit of the B-family polymerase Pol ζ on one face, while simultaneously binding the regulatory subunit Rev7 of Polζ on the opposite face. − …”

“…6 The Y-family polymerases Rev1, Polη, Polι, and Polκ and the B-family polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) are responsible for the replicative bypass of the majority of DNA lesions in the process of Rev1/Polζ-dependent TLS. 7,8 Rev1 is a Y-family DNA polymerase with limited deoxycytidyltransferase activity 9 whose primary function is to serve as a scaffold for assembly of the multiprotein TLS complex and regulate recruitment of other TLS polymerases through multiple protein−protein interactions (PPIs). 7,8 Most of these essential PPIs with other TLS DNA polymerases are mediated through the C-terminal domain of Rev1 (Rev1-CT).…”

Stalling of Eukaryotic Translesion DNA Polymerases at DNA-Protein Cross-Links

“…To investigate the ability of hPOLι to approach a DPC, we performed primer extension reactions on the model DPC-containing substrates in the presence of dNTPs and Mg 2+ or Mn 2+ ions ( Figure 2 a). hPOLι is a low-processivity DNA polymerase, typically incorporating 1–3 nucleotides per association [ 46 ]. However, it is more active on gapped DNA substrates incorporating 7–10 nucleotides and possesses limited strand displacement activity [ 47 , 48 ].…”

Targeting the Translesion Synthesis Pathway for the Development of Anti-Cancer Chemotherapeutics

“…Nevertheless, there is also a significant diversity in active site structures and detailed mechanisms of TLS polymerases, with different TLS enzymes specialized on a bypass of distinct types of DNA damage (cognate lesions). ,,− The prominent example is accurate and efficient bypass of the most common UV-induced DNA lesion, TT cyclobutane pyrimidine dimers (TT-CPDs), by polη. − TT-CPDs can also be copied over by other TLS enzymes in a less accurate, more mutagenic manner; , therefore, loss of polη activity in xeroderma pigmentosum variant (XPV) patients results in hypersensitivity to sunlight and predisposition to skin cancer. , Additionally, polη is proficient in nucleotide incorporation opposite intrastrand G–G cross-links caused by cisplatin (cisPt-GG), ,− and a number of other lesions . Another Y-family TLS polymerase, polκ, can also insert nucleotides opposite various lesions, as well as extend the aberrant DNA primer terminus; , most notably, it can efficiently and accurately bypass N 2 -dG adducts such as those formed by benzo­[ a ]­pyrene (BaP-G) present in tobacco smoke. − Polι is the least accurate TLS enzyme related to polη, ,, which uniquely utilizes Hoogsteen pairing of the incoming nucleotides with the template bases and often misincorporates dGMP opposite T bases. , Catalytic activity of Rev1 is limited to dCMP incorporation across G-templates and abasic sites, − while the B-family polymerase polζ primarily specializes in extension of the distorted primer-template following sites of DNA damage, − ,, but can also incorporate nucleotides across certain DNA lesions. − …”

“…6 Another Y-family TLS polymerase, polκ, can also insert nucleotides opposite various lesions, 6 as well as extend the aberrant DNA primer terminus; 78,79 most notably, it can efficiently and accurately bypass N 2 -dG adducts such as those formed by benzo[a]pyrene (BaP-G) present in tobacco smoke. 80−82 Polι is the least accurate TLS enzyme related to polη, 23,24,83 which uniquely utilizes Hoogsteen pairing of the incoming nucleotides with the template bases and often misincorporates dGMP opposite T bases. 84,85 Catalytic activity of Rev1 is limited to dCMP incorporation across G-templates and abasic sites, 86−90 while the B-family polymerase polζ primarily specializes in extension of the distorted primer-template following sites of DNA damage, [33][34][35][36]53,54 but can also incorporate nucleotides across certain DNA lesions.…”