Lesion Bypass Activity of DNA Polymerase θ (POLQ) Is an Intrinsic Property of the Pol Domain and Depends on Unique Sequence Inserts

Abstract

POLQ (polθ) is a large, multi-domain DNA polymerase0020encoded in higher eukaryotic genomes. It is important for maintaining genetic stability in cells and helping protect cells from DNA damage caused by ionizing radiation. POLQ contains an N-terminal helicase-like domain, a large central domain of indeterminate function, and a C-terminal polymerase domain with sequence similarity to the A-family of DNA polymerases. The enzyme has several unique properties, including low fidelity and the ability to insert and extend past abasic sites and thymine glycol lesions. It is not known whether the abasic site bypass activity is an intrinsic property of the polymerase domain, or whether helicase activity is also required. Three “insertion” sequence elements present in POLQ are not found in any other A-family DNA polymerase and it has been proposed that they may lend some unique properties to POLQ. In this work we analyzed the activity of the DNA polymerase in the absence of each sequence insertion. We find that the pol domain is capable of highly efficient bypass of abasic sites in the absence of the helicase-like or central domains. Insertion 1 increases the processivity of the polymerase but has little, if any, bearing on the translesion synthesis properties of the enzyme. However, removal of insertions 2 and 3 reduces activity on undamaged DNA and completely abrogates the ability of the enzyme to bypass abasic sites or thymine glycol lesions.

Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids

“…Polθ has multiple documented activities in DNA replication and repair, including alt-EJ, replication repair, translesion synthesis, and replication initiation (4)(5)(6)(7)(8)10,15,31,32). Although the activities and cellular functions of Polθ-polymerase have been investigated, little is understood regarding the enzymatic activities of Polθ-helicase.…”

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

“…POLQ depends on unique sequence inserts that are also required for polymerization proficiency and processivity (55). Thus, we constructed a TPR2 deletion mutant of B35DNAP (ΔTPR2; Materials and Methods), which, as was foreseen for all proteinprimed DNA polymerases (23), has impaired activities (Fig.…”

A Role for DNA Polymerase θ in Promoting Replication through Oxidative DNA Lesion, Thymine Glycol, in Human Cells

“…Unlike prokaryotic A-family polymerases and unlike even the eukaryotic pol homolog Mus308 in Drosophila, human and mouse Pol harbor insertion elements 1, 2, and 3, in the polymerase domain. The effects of deletions of these insertion elements on DNA synthesis have been examined in a protein comprised of residues 1792-2590, which lacks the DNA helicase and central domains but retains the DNA polymerase domain and DNA synthesis activity (35). Any further shortening of the N-terminal portion, however, inactivates polymerase function (35).…”

“…The effects of deletions of these insertion elements on DNA synthesis have been examined in a protein comprised of residues 1792-2590, which lacks the DNA helicase and central domains but retains the DNA polymerase domain and DNA synthesis activity (35). Any further shortening of the N-terminal portion, however, inactivates polymerase function (35). The removal of insertion loops 2 or 3 results in a considerable reduction in the ability of the pol-(1792-2590) protein to synthesize DNA; the removal of insertion loop 1, however, has much less of an adverse effect on DNA synthesis (35).…”