The Pol \u03b2 variant containing exon \u03b1 is deficient in DNA polymerase but has full dRP lyase activity

Da-Peng Dai,Samuel H Wilson,Rajendra Prasad,Phyllis R Strauss

doi:10.1038/s41598-019-45846-0

Abstract

DNA polymerase (Pol) β is a key enzyme in base excision repair (BER), an important repair system for maintaining genomic integrity. We previously reported the presence of a Pol β transcript containing exon α (105-nucleotide) in normal and colon cancer cell lines. The transcript carried an insertion between exons VI and VII and was predicted to encode a ~42 kDa variant of the wild-type 39 kDa enzyme. However, little is known about the biochemical properties of the exon α-containing Pol β (exon α Pol β) variant. Here, we first obtained evidence indicating expression of the 42 kDa exon α Pol β variant in mouse embryonic fibroblasts. The exon α Pol β variant was then overexpressed in E. coli, purified, and characterized for its biochemical properties. Kinetic studies of exon α Pol β revealed that it is deficient in DNA binding to gapped DNA, has strongly reduced polymerase activity and higher Km for dNTP during gap-filling. On the other hand, the 5′-dRP lyase activity of the exon α Pol β variant is similar to that of wild-type Pol β. These results indicate the exon α Pol β variant is base excision repair deficient, but does conduct 5′-trimming of a dRP group at the gap margin. Understanding the biological implications of this Pol β variant warrants further investigation.

Highlights

DNA polymerase β is the smallest naturally occurring cellular DNA polymerase and is found as a single polypeptide chain of ~39 kDa with only 335 amino acid residues[1,2]
During SP base excision repair (BER), these domains are responsible for removal of the 5′-sugar phosphate group and template directed insertion of a dNMP into the DNA gap produced by AP endonuclease 1 (APE1) incision of the AP-site containing BER intermediate[7,8,15,19,20]
The results revealed that the recombinant exon α Pol β variant co-migrated in the gel with the endogenous larger Pol β polypeptide (Fig. 3B)

Summary

Introduction

DNA polymerase β is the smallest naturally occurring cellular DNA polymerase and is found as a single polypeptide chain of ~39 kDa with only 335 amino acid residues[1,2]. Using the reverse transcriptase-polymerase chain (RT-PCR) assay, we and others found variant transcripts of the Pol β mRNA in human cell lines: A 58-bp deletion of exon II; and a 87-bp deletion of exon XI22–24. In addition to these splicing isoforms, we observed a variant with a 107-bp insertion between exons IX and X, and this variant was reported by another research group at the same time[25]. We report that the recombinant form of the 42 kDa exon α Pol β variant was overexpressed in E. coli, purified and characterized for its BER properties

Methods

Results

Conclusion