LMG speaker discusses replication beyond DNA damage
By Jeffrey Stumpf
After low levels of DNA damage, cells may not need a large gene expression response to complete DNA replication, according to research presented Nov. 19 at NIEHS by Kenneth Marians, Ph.D.
Marians, dean of the Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, described clear evidence that DNA replication, by itself, can restart beyond a replication blocking DNA lesion. His research suggests that DNA replication is more tolerant of DNA damage than previously thought.
Many environmental agents, such as ultraviolet (UV) light, damage DNA by causing roadblocks that stall DNA polymerases. All forms of life have evolved methods of dealing with DNA damaging agents, including removing the damaged base; recruiting a more tolerant polymerase that bypasses the lesion; providing a detour for the polymerase using recombination or switching the template; and signaling for cell death via apoptosis.
Disruption to these pathways are directly linked to genetic or environmentally-induced predisposition to cancer. Inducing the proteins that promote the DNA damage response pathways is costly to the cell. Marians argues that the replication complex, or replisome, may be able to deal with small amounts of DNA damage by skipping past the damage.
“I think that replisome skipping is a housekeeping function that is there to take care of background levels of damage and not induce DNA damage responses,” Marians postulated. “There’s no reason to induce the response if there is only a couple of lesions in the DNA.”
E. coli proteins mind the gap to finish what they started
The well-accepted dogma of nuclear and bacterial DNA replication states that continuous leading strand DNA replication is coupled to discontinuous lagging strand replication. While the lagging strand restarts with every Okazaki fragment, Marians asked whether leading strand replication could also replicate discontinuously past a DNA lesion.
His research took advantage of the ability to reconstitute the E. coli replisome, to demonstrate replication past a carefully made DNA template containing a cyclobutane pyrimidine dimer, a common lesion caused by exposure to UV light. Using this system, Marians showed that the replisome restarts replication several hundred nucleotides past a lesion, or consecutive lesions, creating a small gap that would be repaired by special gap filling proteins in vivo. Interestingly, Marians demonstrated that restarted replication occurred with the same kinetics as replication prior to the lesion.
Switching polymerases in mid-replication
Though discontinuous leading strand synthesis by the replisome leaves a small gap at a DNA lesion, synthesizing DNA without a gap requires translesion synthesis (TLS) by a specialized polymerase. In E. coli, DNA polymerase IV (Pol IV) performs TLS by gaining access to the replication fork and inserting a nucleotide opposite the DNA lesion.
Marians used his replication system to understand how the normal replisome hands off the replication fork to Pol IV. He showed that TLS by Pol IV requires the same DNA tethering protein complex, called the beta clamp, as the normal replisome. Thus, the beta clamp may function as a tool belt that provides an adequate polymerase to the replication fork quickly.
Humans contain many TLS polymerases and a similar DNA tethering protein, suggesting that a similar mechanism may be important for human DNA replication. Mutations in the gene that encodes the TLS DNA polymerase eta cause a variant of xeroderma pigmentosum, which leads to an extreme sensitivity to light and a high risk of skin cancer. By understanding TLS mechanisms, Marians’ work will lead to a better understanding for how polymerase switching is important to resisting UV damage and preventing cancer.
(Jeffrey Stumpf, Ph.D., is a research fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group and a frequent contributor to the Environmental Factor.)