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. Author manuscript; available in PMC: 2020 Jan 10.
Published in final edited form as: Cell. 2019 Jan 10;176(1-2):127–143.e24. doi: 10.1016/j.cell.2018.12.008

Figure 7. E. coli DNA Pol IV, Human DNMT1 Promote DNA Damage via Binding the Replisome Clamp.

Figure 7.

(A) E. coli Pol IV in cluster with high DNA loss.

(B) Left: Pol IV promotion of DNA damage is reduced by overproducing its competitor Pol II. Right: Pol IV promotion of DNA damage requires interaction with beta (β) replisome sliding clamp—ΔBBD, deleted β-binding domain—and is partly independent of Pol IV catalytic activity (cat− mutant).

(C) Mean ± SEM of ≥3 experiments.

(D) DNA damage reduced by reduction of Pol IV-β interaction with tau-only mutant, which favors Pol III. RecB- and RecF-dependence of Pol IV-induced DNA damage implicate DSBs and single-strand gaps.

(E) Mean ± SEM of ≥3 experiments. Pol IV is induced by IPTG.

(F) Model: Pol IV induces DNA damage by excess binding the β clamp. Excess β interaction might slow the replisome causing fork breakage/collapse, or displace β-binding DNA-repair proteins, among other possibilities. 8-oxo-dG-independence, Figure S7F,G.

(G) Mutant derivatives of human DNA methyltransferase DNMT1 (WT, wild-type). PBD, PCNA-binding domain; U, UHRF1 (ubiquitin-like PHD and RING-finger 1 interacting domain; RFTS, (recruits DNMT1 to DNA-methylation sites); Cat and C1226A, catalytically inactivate mutants, all N-terminally GFP tagged.

(H) Human DNMT1 overproduction in human cells promotes γH2AX accumulation methylase-independently and replisome-clamp-interaction dependently.

(I) Elevated DNMT1 promotes PCNA monoubiquitination (replication-stress) replisome-interaction dependently. Western blot with anti-PCNA antibody.

(J) Model/hypotheses for how excess DNMT1 promotes DNA damage.

(K) Hypothesis: DDPs, a cancer-protein function class upstream of DNA repair. Excessive endogenous DNA damage could titrate (thick blue -|) or inhibit (thin black -|) DNA repair causing DNA-repair-protein deficiency without a DNA-repair-gene mutation. Repair deficiency increases mutation rate, and cancer- (or evolution-) driving mutations in cell-biology-altering “gatekeeper” genes that cause the cancer phenotypes.