Stop Stalling: Mus81 Required for Efficient Replication
DNA combing demonstrates that Mus81 affects DNA replication rates.
DNA replication is precisely controlled to ensure that daughter cells receive intact, accurate genetic information. Each segment of DNA must be copied only once, and the rate of replication coordinated genome-wide. Mild replication stress slows DNA synthesis and activates a pathway involving the Mus81 endonuclease, which generates a series of DNA breaks that are rapidly repaired, allowing the cell to avoid activating the S-phase checkpoint and its potentially damaging outcomes of apoptosis or error-prone repair. Mirit Aladjem, Ph.D., of CCR’s Developmental Therapeutics Branch, and her colleagues wondered whether Mus81 also plays a role in regulating the replication rate during growth in the absence of stress.
The researchers began their studies by measuring DNA replication fork progress in wild type or Mus81 knockout (Mus81-/-) HCT116 colorectal cancer cells. They labeled cells sequentially with the thymidine analogs iodo-deoxyuridine (IdU) and chloro-deoxyuridine (CldU), harvested DNA fibers, mounted them on slides, and visualized IdU and CldU incorporation with immunofluorescence as green and red tracks, respectively. The investigators found that, even though both cell types had similar cell cycle profiles, Mus81-/- cells had shorter replication tracks during normal cell growth, indicating significantly slower replication. This reduced average rate could be due to mild uniform reduction in DNA synthesis or to significant reduction in synthesis at a subset of sites.
Using the same sequential labeling technique, the scientists next measured the distance between replication origins to determine whether there was a change in the frequency of initiation events. Lack of Mus81 resulted in shorter average inter-origin distances, indicating more frequent replication initiation than wild type cells. The shorter observed replication tracks could be due to a slower rate of DNA synthesis or to replication fork stalling. These alternatives can be distinguished by measuring replication fork asymmetry, that is, replication origins that have a longer track on one side than the other, which is caused by stalling. Cells expressing or lacking Mus81 showed similar rates of asymmetric replication tracks, supporting the idea that slower DNA synthesis in the absence of Mus81 shortens the tracks. The researchers found a similarly reduced replication rate and increased replication frequency in HCT116 cells and MDA-MB-231 breast cancer cells treated with small interfering RNAs (siRNAs) to acutely reduce Mus81 levels, suggesting these are bone fide phenotypes of Mus81 loss. Massively parallel sequencing of newly replicated DNA strands revealed that wild type and Mus81 knockout cells initiate replication at similar sites.
Because Mus81 is a structure-specific endonuclease, the investigators wondered whether its enzymatic activity was necessary for regulating the rate of DNA synthesis. They mutated two critical aspartic acid residues within the nuclease domain to alanines to abolish activity and expressed the double mutant or wild type Mus81 in the Mus81-/- cells. While neither protein affected cell cycle progression, intact Mus81 partially rescued the slow replication of the knockout cells. In contrast, mutant Mus81 slowed replication and reduced inter-origin distances, implicating the endonuclease activity of Mus81 in regulating the rate of DNA replication.
Mus81 induces DNA breaks at stalled replication forks to avoid checkpoint activation and allow replication to resume. This suggested to the scientists that there might be a difference in the number of spontaneous DNA breaks in cells with and without Mus81 expression. However, DNA breaks were similar or even slightly lower in cells lacking Mus81 compared to wild type cells. To establish whether Mus81 endonuclease activity is important for recovery from S-phase stress, the researchers treated wild type and Mus81-/- cells with non-toxic doses of the DNA polymerase alpha inhibitor aphidicolin (APH) and measured double-strand breaks. Cells expressing Mus81 showed high levels of double-strand breaks while there was no significant change with or without APH in cells lacking Mus81. Colony formation assays, in contrast, revealed that Mus81-/- cells were more sensitive to APH, suggesting the DNA breaks generated by Mus81 promoted cell survival by facilitating replication fork recovery. In fact, cells lacking Mus81 completely arrested replication when treated with APH whereas replication continued at a slow pace in Mus81-expressing cells. The endonuclease activity of Mus81 was required since the nuclease domain double mutant was unable to rescue replication when expressed in Mus81-/- cells treated with APH. Data from bromo-deoxyuridine (BrdU) comet assays revealed that slower replication forks in cells lacking Mus81 are not due to altered processing of the short DNA pieces formed at the lagging strand during DNA replication, known as Okazaki fragments.
XPF is another endonuclease in the same family as Mus81 and may compensate for Mus81 during normal growth. To test this idea, the investigators treated wild type and Mus81-/- HCT116 cells with siRNAs against XPF. Loss of XPF in both cell types reduced DNA synthesis, but dual loss of Mus81 and XPF did not synergistically enhance the effect. In terms of survival, however, cells lacking both Mus81 and XPF had a higher percentage of apoptotic cells than those without Mus81 alone. Likewise, loss of XPF in wild type HCT116 cells reduced survival, but the effect was stronger in Mus81-/- cells. Thus, Mus81 and XPF seem to work together to prevent DNA lesions during normal growth, but loss of both leads to cell death.
Together, these results demonstrate that Mus81 helps regulate the rate of DNA synthesis under normal growth conditions and in the presence of drugs that slow replication. The endonuclease activity of Mus81 is required in both cases, suggesting the role of Mus81 is to resolve low-frequency lesions.Summary Posted: Wed, 04/01/2015
Fu H, Martin MM, Regairaz M, Huang L, You Y, Lin CM, Ryan M, Kim RG, Shimura T, Pommier Y, and Aladjem M. The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage. Nature Communications. April 16, 2015 PubMed Link