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. 2016 Sep 6;44(15):7242-50.
doi: 10.1093/nar/gkw439. Epub 2016 May 16.

In Vivo Evidence for Translesion Synthesis by the Replicative DNA Polymerase δ

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Free PMC article

In Vivo Evidence for Translesion Synthesis by the Replicative DNA Polymerase δ

Kouji Hirota et al. Nucleic Acids Res. .
Free PMC article

Abstract

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.

Figures

Figure 1.
Figure 1.
pold1exo- mutation restores mutant phenotype of pold3 cells. (A) pold1exo- mutation significantly suppresses the growth defect of pold3 cells. The doubling time for the indicated genotypes is indicated. Error bars represent standard deviations (SD) from three independent assays. Statistical significance was determined by a Student's t-test and P-value was calculated. (*) P < 0.01, (**) P < 0.001. (B) Expression of POLD1exo− reverses the synthetic lethality of polη/polζ/pold3 cells. Growth curves of the indicated cells are shown after addition of doxycycline at time zero. The tet-POLD3 transcription was active without doxycyclin (ON) and inhibited upon addition of doxycyclin (OFF). (C) Expression of POLD1exo− reverses sensitivities of pold3 cells to MMS and H2O2. Indicated cells were exposed to MMS or H2O2. The dose of the genotoxic agent is displayed on the x-axis on a linear scale, while the percentage fraction of surviving cells is displayed on the y-axis on a logarithmic scale. Error bars show the SD for three independent assays. Statistical significance was determined by a Student's t-test and P-value was calculated. (*) P < 0.01.
Figure 2.
Figure 2.
Expression of the proof reading exonuclease deficient Polδ substantially changes the mutation spectrum of the Ig Vλ hypermutation. (A) Ig Vλ segments isolated from indicated cells, clonally expanded for two weeks. Horizontal lines represent the rearranged Ig Vλ (450 bp), with hypermutation (lollipop shapes), single-nucleotide substitutions that could be the result of hypermutation or gene conversion (vertical bars). At least three cellular clones were expanded for two weeks and analyzed for each data set. (B) The rates of TLS-dependent hypermutation (PM) are indicated with standard error. White bars represent the rate of G/C to A/T mutations, TLS following A-rule. Data from polη/polζ cells are taken from (19) for comparison. Statistical significance was determined by a Fishers exact test and P-value was calculated. (*) P < 0.05. (C) Pattern of point mutation in wild type, pold3, pold1exo and pold3/pold1exo- cells. Tables showing the pattern of mutation in each line. Some of data for wild type, pold3 and polη/polζ cells are from (19).
Figure 3.
Figure 3.
Expression of POLD1exo− restores defective replication fork progression past UV damaged DNA in pold3 cells. (A) Schematic for DNA fiber labelling. DT40 cells were labeled sequentially with IdU and CldU with or without UV treatment after IdU labeling. The right hand panel shows an example of an ongoing fork. The arrowhead indicates the direction of replication. (B) The data for cells carrying the indicated genotypes was plotted as a cumulative percentage (y-axis) of forks at each ratio (x-axis). The transcription of tet-POLD3 was repressed by doxycycline for 1 day. The P-values of the Kolmogorov-Smirnov test for ratio distribution of each mutant for UV compared to wild type are indicated. n.s.: not significant. A part of data for pold3 and polη/polζ/pold3 cells were from (19).
Figure 4.
Figure 4.
Expression of POLD1exo− changes the mutation spectrum of TLS past CPD. (A) A CPD placed opposite GpC mismatch was randomly integrated into the genome using the PiggyBlock vector. TLS across the CPD results in a dual peak in the resulting cellular clone (left), while template switching results in a homogenous GC read (right). (B) The pattern of nucleotide incorporation opposite the CPD site. The percentage of each nucleotide incorporated at each position is indicated by the size of the letter of the nucleotide in the column. The total numbers of TLS events are shown. (C) A schematic representation of the opposed arrangement of CPD photoproducts in the piggyBlock plasmid (piggyBlock-op) and possible outcomes of DNA replication, only by TLS, over the lesion. (D) The pattern of nucleotide incorporation opposite the CPD in polη/polζ/xpa and polη/polζ/xpa/pold1exo− cells in piggyBlock-op plasmid.
Figure 5.
Figure 5.
Inactivation of proofreading activity significantly increases the efficiency of Polδ (POLD3+) to perform TLS past abasic sites and UV damage. (A) DNA synthesis reactions were carried out with 2 nM of proofreading proficient (WT) or deficient (exo) Polδ holoenzyme for the indicated duration. The histogram shows amounts of the fully extended product at the indicated time points. Error bars show the SD for three independent assays. (B) DNA synthesis reactions carried out with the indicated Polδ holoenzymes on template and primer strands, which are schematically shown on the left. Amount of the fully extended product was analyzed at 15 min. Error bars show the SD for three independent assays. (C) The pie charts indicate percentage of the nucleotides inserted opposite abasic site.

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