DNA polymerase mu (Pol micro), homologous to TdT, could act as a DNA mutator in eukaryotic cells

Abstract

A novel DNA polymerase has been identified in human cells. Human DNA polymerase mu (Pol mu), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT). Human Pol mu, overproduced in Escherichia coli in a soluble form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn(2+) ions. Interestingly, unlike TdT, the catalytic efficiency of polymerization carried out by Pol mu was enhanced by the presence of a template strand. Using activating Mg(2+) ions, template-enhanced polymerization was also template-directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values. In the presence of Mn(2+) ions, template-enhanced polymerization produced a random insertion of nucleotides. Northern-blotting and in situ analysis showed a preferential expression of Pol mu mRNA in peripheral lymphoid tissues. Moreover, a large proportion of the human expressed sequence tags corresponding to Pol mu, present in the databases, derived from germinal center B cells. Therefore, Pol mu is a good candidate to be the mutator polymerase responsible for somatic hyper- mutation of immunoglobulin genes.

DNA polymerases in adaptive immunity

“…In the yeast Saccharomyces cerevisiae, the Pol4 enzyme, which belongs to the PolX family, has been shown to be involved in fill-in DNA synthesis during NHEJ 14 . Among PolX polymerases, Polλ is closer to yeast Pol4 while Polµ and TdT, which are highly homologous to each other, are more distantly related [15][16][17] .…”

Transcription, β–like DNA polymerases and hypermutation

“…In contrast, the group of Honjo identi¢ed an activity acting probably upstream in the centroblast di¡erentiation pathway (see Muramatsu et al 2000). A second argument is the remarkable in¢delity of Pol m reported by the group of Blanco (Dominguez et al 2000), even though several translesion DNA polymerases (eta and iota, in particular) appear competitive in this respect (Matsuda et al 2000;Tissier et al 2000). Finally, mainly its relatedness with TdT is the most appealing feature for the immunologistö two enzymes would have evolved in higher eukaryotes: TdT, to generate diversity of CDR3 by random nucleotide insertion at the V^D^J junction; and a TdT-like polymerase with a poor ¢delity to diversify the whole V gene at later di¡erentiation steps.…”

“…We report here the identi¢cation of two new b-like enzymes, one of which (Pol m), isolated in parallel by the group of Luis Blanco (Dominguez et al 2000), has a preferred lymphoid expression pattern and is closely related to terminal deoxynucleotidyl transferase (TdT), an enzyme whose unique function is the diversi¢cation of the T-and B-cell repertoire.…”

DNA polymerases in nonhomologous end joining: Are there any benefits to standing out from the crowd?

“…Pol l was identified as a 55-kDa protein with high homology (identity, 41%) to TdT [Dominguez et al, 2000]. Pol l can extend unpaired primer termini by template-independent addition [Dominguez et al, 2000] though this activity is much lower than observed with TdT.…”

“…Pol l can extend unpaired primer termini by template-independent addition [Dominguez et al, 2000] though this activity is much lower than observed with TdT. Unlike TdT, Pol l is most active when it can add nucleotides complementary to a template, and when it can recognize the 5 0 -phosphate terminus of a downstream strand associated with this template [Dominguez et al, 2000]. Together with Pol l's ability to form a complex with the NHEJ core factors Ku and XRCC4-ligase IV and take advantage of their end aligning activity, Pol l can promote NHEJ of noncomplementary ends (Fig.…”