Cellular Roles of DNA Polymerase Beta

Since its discovery and purification in 1971, DNA polymerase ß (Pol ߆) is one of the most well-studied DNA polymerases. Pol ß is a key enzyme in the base excision repair (BER) pathway that functions in gap filling DNA synthesis subsequent to the excision of damaged DNA bases. A major focus of our studies is on the cellular roles of Pol ß. We have shown that germline and tumor-associated variants of Pol ß catalyze aberrant BER that leads to genomic instability and cellular transformation. Our studies suggest that Pol ß is critical for the maintenance of genomic stability and that it is a tumor suppressor. We have also shown that Pol ß functions during Prophase i of meiosis. Pol ß localizes to the synaptonemal complex and is critical for removal of the Spo11 complex from the 5’ ends of double-strand breaks. Studies with Pol ß mutant mice are currently being undertaken to more clearly understand the function of Pol ß during meiosis. in this review, we will highlight our contributions from our studies of Pol ß germline and cancer-associated variants

Heterozygosity for a hypomorphic polβ mutation reduces the expansion frequency in a mouse model of the fragile x-related disorders

Author Summary Unstable microsatellites are responsible for a number of debilitating human diseases known as the Repeat Expansion Diseases. The unstable microsatellites, which consist of tandem arrays of short repeat units, are prone to increase in length (expand) on intergenerational transmission and during the lifetime of the individual. Unlike the typical microsatellite instability seen in disorders like Lynch syndrome that arise from mutations in mismatch repair (MMR) genes, expansions of these microsatellites are abolished when MMR is lost. However, how MMR, which normally protects the genome against microsatellite instability, actually promotes microsatellite expansions in these diseases is unknown. There is evidence to suggest that a second DNA repair process, base excision repair (BER), may be involved, but whether the nicks generated early in the BER-process are subverted by an MMR-dependent pathway that generates expansions or whether some MMR proteins contribute to a BER-based expansion process is unclear. Here we show that a mutation that reduces the activity of Polβ, an essential BER enzyme, also reduces the expansion frequency. Since Polβ is essential for key events in BER downstream of the generation of nicks, our data favor a model in which expansions occur via a BER-dependent pathway in which MMR participates

The effect of heterozygosity for the <i>PolBC</i> mutation on the number of expansions, contractions and unchanged alleles seen in the gametes of 3-month-old male mice.

<p>Small pool PCR was carried out on sperm DNA isolated from two 3-month-old <i>PolB+/+</i> and two 3-month-old <i>PolB+/C</i> male mice as described in the Materials and Methods. These animals all had ~140 repeats. The difference between the number of expansions, contractions and unchanged alleles within each genotype and between the two genotypes was evaluated by Fisher’s exact test. The error bars represent the 95% confidence intervals. There were no significant within genotype differences in the frequency of expansions, contractions or unchanged alleles. Allele classes that are significantly different in <i>PolB+/C</i> mice are marked with asterisks. Expansions were significantly reduced in <i>PolB+/C</i> gametes (p = 0.0001) and contractions significantly increased (p = 0.0001) by Fisher’s exact test.</p

PolB+/C mice show a reduced somatic instability index in testis and tail.

<p>The somatic instability index of different organs of three 16 month old <i>PolB+/+</i> and three 16 month old <i>PolB+/C</i> mice with ~140 repeats was determined as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005181#pgen.1005181.ref046" target="_blank">46</a>]. Tail 1 and tail 2 refer to tail samples taken at 3 weeks of age and tail samples taken at 16 months respectively. The error bars represent the standard deviations. The significance of the differences in the SII for different genotypes was determined using Student’s t-test. The tissues in which the SII was significantly lower in <i>PolB+/C</i> mice are indicated by asterisks. The SII for <i>PolB+/C</i> testis was significantly lower at p = 0.001 and the SII for the <i>PolB+/C</i> tail 2 sample was significantly lower at p = 0.013.</p

The effect of heterozygosity for the <i>PolBC</i> mutation on the distribution of repeat number changes seen in the gametes of 3-month-old male mice.

<p>The percentage of alleles with the indicated gains or losses in repeat number for <i>PolB+/+</i> and <i>PolB+/C</i> mice was plotted. The mean gain of repeats was 3.17 (SD = 2.52) for <i>PolB+/+</i> and 5.43 (SD = 4.48) for <i>PolB+/C</i>. This resulted in a distribution of expanded alleles that was significantly different in the two genotypes (p = 0.0001; <i>t</i> test). The mean loss of repeats was 10.83 (SD = 11.05) for <i>PolB+/+</i> and 19.68 (SD = 31.58) for <i>PolB+/C</i>. The very high standard deviations due to the presence of some very large contractions particularly in the <i>PolB+/C</i> mice resulted in a distribution of contracted alleles that was not significantly different in the two genotypes. Inset: <i>PolB+/C</i> mice have fewer small expansions and more large expansions than <i>PolB+/+</i> mice. The error bars represent the 95% confidence interval. Repeat size classes that are significantly different in <i>PolB+/C</i> mice are marked with an asterisk. The decrease in the number of alleles with 1–5 repeats was significant at p = 0.0001, and the increase in the number of alleles with >10 repeats was significant at p = 0.005.</p

Expression of various BER proteins in different mouse organs.

<p>Total protein was extracted from different organs of 3 different FXD mice as described in the Materials and Methods. Since in our experience proteins used as “normalizing controls” including β-actin and α-tubulin and GAPDH differ significantly in different organs, we took care to analyze equal amounts of protein as assessed by the Bradford Assay. Ten micrograms of protein from the organs of each animal were pooled and loaded onto 3–8% Tris-Acetate gels, resolved by gel electrophoresis and subjected to Western blotting as described in the Materials and Methods.</p

The effect of heterozygosity for the <i>PolBC</i> mutation on the distribution of repeat number changes seen in the gametes of 11-month-old male mice.

<p>The percentage of alleles with the indicated change in repeat number that were seen in the gametes of three 11-month-old mice <i>PolB+/+</i> and three 11-month old <i>PolB+/C</i> mice. The grey arrowheads indicate the local maxima seen in the distribution of <i>PolB+/C</i> alleles. The mean gain of repeats was 8.75 (SD = 5.96) for <i>PolB+/+</i> and 14.04 (SD = 8.70) for <i>PolB+/C</i>. This resulted in a distribution of expanded alleles that was significantly different in the two genotypes (p = 0.0001; <i>t</i> test). Too few contractions were seen to carry out any statistical analysis. Inset: <i>PolB+/C</i> mice have fewer small expansions and more large expansions than <i>PolB+/+</i> mice. The error bars represent the 95% confidence interval. Repeat size classes that are significantly different in <i>PolB+/C</i> mice are marked with an asterisk. The decrease in the number of alleles with 1–5 repeats was significant at p = 0.0001, and the increase in the number of alleles with >15 repeats was also significant at p = 0.0001.</p

Model for BER-mediated repeat expansion in the FX PM mouse.

<p>Nicks that do not get repaired by short patch BER may be channeled into one of two branches of the LP BER pathway. (<i>i) The Polβ-dependent</i>, <i>Polδ/Polε-independent branch</i>. Nick processing is carried out by Polβ, a poorly processive polymerase with weak strand-displacement activity. The resultant small flaps are processed by FEN1 to generate a ligatable 5’ end that still contains a few additional flap bases (shown in orange). (ii) The <i>Polβ/Polδ/Polε-dependent branch</i>. Since both Polδ and Polε are more processive than Polβ, more strand slippage and more extensive strand displacement may result. Repriming of DNA synthesis on the slipped-strand using Polβ would not remove looped out bases (shown in red) since Polβ lacks a suitable proofreading activity. Limited strand displacement by Polβ or more extensive strand displacement by Polδ/Polε, followed by FEN1 cleavage could also result a ligatable end that still contains some flap bases (shown in orange). In either case, repair synthesis initiated on the complementary strand would fix the supernumerary bases into the derivative allele thus generating either a small (i) or large (ii) expansion. MMR proteins may facilitate this process by stabilizing the hairpins. These proteins may also directly generate expansions by channelling the hairpins formed during BER into the MMR pathway.</p