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Human DNA polymerase ι (polι) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that polι is able to facilitate limited translesion replication of a thymine–thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that polι misinserts T or G opposite the 3′ T of the CPD ∼1.5 times more frequently than the correct base, A. While polι is unable to extend the T·T mispair significantly, the G·T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A·T base pair. By comparison, polι readily misinserts two bases opposite a 6‐4 thymine–thymine pyrimidine–pyrimidone photoproduct (6‐4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that polι possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.

A hallmark of human DNA polymerase ι (polι) is the asymmetric fidelity of replication at template A and T when the enzyme extends primers annealed to a single‐stranded template. Here, we report on the efficiency and accuracy of polι‐dependent replication at a nick, a gap, the very end of a template and from a mispaired primer. Polι cannot initiate synthesis on a nicked DNA substrate, but fills short gaps efficiently. Surprisingly, polι's ability to blunt‐end a 1 bp recessed terminus is dependent upon the template nucleotide encountered and is highly erroneous. At template G, both C and T are inserted with roughly equal efficiency, whilst at template C, C and A are misinserted 8‐ and 3‐fold more often than the correct base, G. Using substrates containing mispaired primer termini, we show that polι can extend all 12 mispairs, but with differing efficiencies. Polι can also extend a tandem mispair, especially when it is located within a short gap. The enzymatic properties of polι appear consistent with that of a somatic hypermutase and suggest that polι may be one of the low‐fidelity DNA polymerases hypothesized to participate in the hypermutation of immunoglobulin variable genes in vivo .

Until recently, the molecular mechanisms of translesion DNA synthesis (TLS), a process whereby a damaged base is used as a template for continued replication, was poorly understood. This area of scientific research has, however, been revolutionized by the finding that proteins long implicated in TLS are, in fact, DNA polymerases. Members of this so-called UmuC/DinB/Rev1/Rad30 superfamily of polymerases have been identified in prokaryotes, eukaryotes and archaea. Biochemical studies with the highly purified polymerases reveal that some, but not all, can traverse blocking lesions in template DNA. All of them share a common feature, however, in that they exhibit low fidelity when replicating undamaged DNA. Of particular interest to us is the Rad30 subfamily of polymerases found exclusively in eukaryotes. Humans possess two Rad30 paralogs, Rad30A and Rad30B. The RAD30A gene encodes DNA polymerase η and defects in the protein lead to the xeroderma pigmentosum variant (XP-V) phenotype in humans. Very recently RAD30B has also been shown to encode a novel DNA polymerase, designated as Pol ι. Based upon in vitro studies, it appears that Pol ι has the lowest fidelity of any eukaryotic polymerase studied to date and we speculate as to the possible cellular functions of such a remarkably error-prone DNA polymerase.