Cloning, purification and characterization of DNA polymerase β from Xenopus laevis

Publisher Website
Google Scholar
Abstract
Double‐strand breaks in the DNA of vertebrate cells are joined by mechanisms of non‐homologous DNA‐end joining (NEJ). In extracts from Xenopus eggs, NEJ is inhibited by dideoxynucleotides, indicating a possible involvement of DNA polymerase β (Polβ). Since some types of NEJ products were shown to be formed in vitro by prokaryotic DNA polymerases lacking exonuclease activity, we were interested in whether Polβ alone would be capable of catalyzing NEJ reactions. Therefore we have cloned the full‐length cDNA of the Xenopus laevis Polβ. The cDNA, predicting a highly conserved 39‐kDa protein of 334 amino acids, was tagged with six histidine residues at its N‐terminus for overexpression in Escherichia coli, purified to near homogeneity, and shown to have the same catalytic properties as the previously cloned rat and human enzymes. Using oligonucleotides as substrates we show that the recombinant Xenopus Polβ adds single untemplated nucleotides to blunt ends. However, under conditions that permit efficient NEJ in Xenopus egg extracts, Polβ does not form those types of NEJ products formed by the prokaryotic polymerases indicating that Polβ alone is not able to mediate the complex NEJ process in vitro. Using substrates with 3′ protruding single strands of increasing length (6−16 nucleotides) we show that Polβ initiates fill‐in DNA synthesis on fold‐back structures formed by the longest 3′ protruding stand. This unusual feature of β‐type polymerases requires that the loop of the fold‐back structure consists of at least six bases and the stem be paired by at least 2bp to facilitate priming of DNA synthesis.

This publication has 0 references indexed in Scilit: