Connecting the Checkpoint with Repair

Abstract

DNA damage can occur through several mechanisms. There can be breakage of the strands or chemical alterations to the DNA, such as alkylation. Double-stranded breaks and alkylation activate different checkpoint pathways, allowing the cell time to repair the damage. Two groups studied the DNA repair and checkpoint signaling activated by alkylation damage in response to methyl methanesulfonate (MMS). Stokes et al. analyzed the requirement for DNA replication during activation of the alkylation checkpoint pathway, using in vitro systems based on Xenopus oocyte extracts or nucleoplasmic extracts. The addition of MMS-treated sperm chromatin to the cycling egg extracts activated the checkpoint kinase ATR, promoting the phosphorylation of one of the ATR substrates Chk-1, and delayed progression through mitosis when compared to egg extracts to which normal sperm chromatin had been added. Activation of ATR and the delayed progression through mitosis were blocked if DNA replication or entry into S phase was inhibited, suggesting that DNA replication may be required for activation of the checkpoint responsible for detection of alkylated DNA. Furthermore, Rad17, a protein that is part of a complex involved in loading the PCNA protein onto replicating DNA, was found to associate specifically with the alkylated DNA and this association required DNA replication. The second group, Hoege et al. , demonstrated that DNA repair involves the PCNA protein and its modification by ubiquitin. In Saccharomyces cerevisiae , PCNA was modified by both ubiquitin and the ubiquitin-related protein SUMO on the same lysine residue. Sumoylation is stimulated during S phase under normal conditions with undamaged DNA. Treatment of the yeast with MMS induced the ubiquitination of PCNA, and this was blocked if genes of the RAD6 repair pathway were mutated. RAD6 is a ubiquitin-conjugating enzyme, which works together with another ubiquitin-conjugating complex, UBC13-MMS2, and the DNA binding RING finger ubiquitin ligases RAD18 and RAD5 in postreplication DNA repair. Genetic interactions occurred between PCNA and these RAD6 pathway genes, altering the ability of the yeast to survive MMS treatment. It will be interesting to see if the Rad17 interaction noted by Stokes et al. and the PCNA modifications reported by Hoege et al. converge on the selective recruitment of differently modified versions of PCNA onto the damaged DNA. M. P. Stokes, R. Van Hatten, H. D. Lindsay, W. M. Michael, DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts. J. Cell Biol. 158 , 863-872 (2002). [Abstract] [Full Text] C. Hoege, B. Pfander, G.-L. Moldovan, G. Pyrowolakis, S. Jentsch, RAD6 -dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419 , 135-141 (2002). [Online Journal]