Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Apr;40:67-76.
doi: 10.1016/j.dnarep.2016.02.002. Epub 2016 Mar 2.

The Role of HERC2 and RNF8 Ubiquitin E3 Ligases in the Promotion of Translesion DNA Synthesis in the Chicken DT40 Cell Line

Affiliations
Free PMC article

The Role of HERC2 and RNF8 Ubiquitin E3 Ligases in the Promotion of Translesion DNA Synthesis in the Chicken DT40 Cell Line

Mohiuddin et al. DNA Repair (Amst). .
Free PMC article

Abstract

The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2(-/-) and RNF8(-/-) cells and HERC2(-/-)/RNF8(-/-) double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2(-/-) and RNF8(-/-) mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks.

Keywords: HERC2; Post-replication repair; RNF8; Translesion DNA synthesis; Ubiquitination.

Conflict of interest statement

Conflict of interest statement

None declared.

Figures

Fig. 1
Fig. 1. Cellular Sensitivity to DNA-damaging agents.
(A–C) Cellular sensitivity to cisplatin (A), UV (B), and MMS (C) was analyzed. Survival rate was calculated as the percentage of surviving cells treated with DNA-damaging agents relative to the untreated surviving cells. The concentration or dose is displayed on the x-axis on a linear scale, while the survival rate is displayed on the y-axis on a logarithmic scale. P-values were calculated by Student’s t-test. Lethal dose 30% (LD30) is the concentration of DNA damaging agents that reduces cellular survival to 30% relative to cells non-treated with DNA damaging agents. LD30 was calculated by the statistics software, R. Error bars show the standard deviation of the mean of at least three independent experiments.
Fig. 2
Fig. 2. The important role of HERC2 and RNF8 in TLS past abasic sites during Ig Vλ hypermutation.
(A) Clones over-expressing AID were expanded for two weeks before isolating genomic DNA and sequencing the Vλ segment. Charts displaying the frequency of each type of mutation. Segment size indicates the proportion of cells in which the number of mutations stated outside the chart had occurred. The total number of sequences analyzed is shown in the centre of the chart. PM = Point mutation/non-templated single base substitution; AMB = Ambiguous; GC = Gene conversion. (B) The rate of point mutation/non-templated single base substitution (PM) events in wild-type cells were set to 1.00 and all values were normalized to the value of wild-type cells and are indicated with standard error. (C) Pattern of point mutation in the indicated cell lines. Tables showing the pattern of mutation in each line, given a percentage of mutations observed.
Fig. 3
Fig. 3. Neither HERC2 nor RNF8 is required for the mono-ubiquitination of PCNA in UV-irradiated cells.
(A) Analysis of ubiquitination of PCNA. Cells were irradiated with 30 J/m2 UV and then incubated for 1 h. Protein extracts were prepared in native conditions for mono-ubiquitination, and analyzed by gel-electrophoresis followed by western blotting using an anti-PCNA antibody. (B) Quantification of ubiquitinated PCNA by immunoblotting. The values for ubiquitinated PCNA were calculated as described in the Materials and Methods. Error bars indicate standard deviation (SD). (C) Analysis of ubiquitination of PCNA in RAD18-/- and RAD18-/-/RAD18-/- cells as in fig. 3A.
Fig. 4
Fig. 4. Intact post-replicative gap filling in HERC2-/- cells.
(A) Schematic diagram of post-replicative gaps filling at UV-damage sites. (B) Indicated cells were irradiated with UV (5 J/m2) and pulse-labelled with [methyl-14C] thymidine for 20 min and either lysed immediately or chased for 3 h in fresh medium containing 10 μM unlabeled thymidine before lysis. Samples were separated on 5–20 % alkaline sucrose gradient sedimentation.
Fig. 5
Fig. 5. HERC2 and RNF8 are dispensable to maintain the velocity of unperturbed DNA synthesis.
(A) Schematic of treatment with UV and pulse labeling with IdU and CldU are shown. Representative image showing stained DNA fibres. (B) Replication fork lengths were obtained by converting the IdU track sizes in µM to kb and analyzed in the indicated cell types. IdU track lengths are calculated by dividing the track lengths by the labeling time and depicted above the track lengths. More than 100 forks were calculated in each cell types. The P-values were calculated by Student’s t-test (C) Indicated cell lines were left untreated and DNA fibres were analyzed for inter-origin distances (IODs). IODs were measured as the distance between two adjacent initiation sites during IdU pulse, and average values are indicated in kb. Over 50 fibers were analyzed in measurement of IODs. The P-values were calculated by Student’s t-test. (D) CldU track lengths were analyzed in the indicated cell types as previous. The P-values for Student’s t-test were *p<0.001. (E) Indicated cell lines were treated with UV and DNA fibres were analyzed for inter-origin distances (IODs) as previous.
Fig. 6
Fig. 6. RNF8 and HERC2 work together to maintain replication fork progression on UV damaged DNA.
(A-C) Replication stalling in response to 20 J/m2 UV (red bars) or sham irradiation (blue bars) in WT (A), HERC2-/-(B) and RNF8-/- (C) cell lines. Each data set is derived from measurement of at least 100 forks. (D) The data for cells carrying the indicated genotypes was plotted as a cumulative percentage (y-axis) of forks at each ratio (x-axis). The P-values of the Kolmogorov–Smirnov test for ratio distribution of each mutant for UV compared to sham treatment are *p<0.002 and **p<0.001.

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

Publication types

Substances

Feedback