Oxidative damage to DNA has been proposed to have a role in cancer and ageing. Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA, and 7, 8-dihydro-8-oxoguanine (8-oxoG) is one of the adducts formed. Eukaryotic replicative DNA polymerases replicate DNA containing 8-oxoG by inserting an adenine opposite the lesion; consequently, 8-oxoG is highly mutagenic and causes G:C to T:A transversions. Genetic studies in yeast have indicated a role for mismatch repair in minimizing the incidence of these mutations. In Saccharomyces cerevisiae, deletion of OGG1, encoding a DNA glycosylase that functions in the removal of 8-oxoG when paired with C, causes an increase in the rate of G:C to T:A transversions. The ogg1Delta msh2Delta double mutant displays a higher rate of CAN1S to can1r forward mutations than the ogg1Delta or msh2Delta single mutants, and this enhanced mutagenesis is primarily due to G:C to T:A transversions. The gene RAD30 of S. cerevisiae encodes a DNA polymerase, Poleta, that efficiently replicates DNA containing a cis-syn thymine-thymine (T-T) dimer by inserting two adenines across from the dimer. In humans, mutations in the yeast RAD30 counterpart, POLH, cause the variant form of xeroderma pigmentosum (XP-V), and XP-V individuals suffer from a high incidence of sunlight-induced skin cancers. Here we show that yeast and human POLeta replicate DNA containing 8-oxoG efficiently and accurately by inserting a cytosine across from the lesion and by proficiently extending from this base pair. Consistent with these biochemical studies, a synergistic increase in the rate of spontaneous mutations occurs in the absence of POLeta in the yeast ogg1Delta mutant. Our results suggest an additional role for Poleta in the prevention of internal cancers in humans that would otherwise result from the mutagenic replication of 8-oxoG in DNA.

The yeast RAD30-encoded DNA polymerase (Pol) bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Human DNA polymerase functions similarly in the bypass of this lesion, and mutations in human Pol result in the cancer prone syndrome, the variant form of xeroderma pigmentosum. UV light, however, also elicits the formation of cis-syn cyclobutane dimers and (6-4) photoproducts at 5-CC-3 and 5-TC-3 sites, and in both yeast and human DNA, UV-induced mutations occur primarily by 3 C to T transitions. Genetic studies presented here reveal a role for yeast Pol in the error-free bypass of cyclobutane dimers and (6-4) photoproducts formed at CC and TC sites. Thus, by preventing UV mutagenesis at a wide spectrum of dipyrimidine sites, Pol plays a pivotal role in minimizing the incidence of sunlight-induced skin cancers in humans.The UV component of sunlight is a major epidemiological risk factor for skin cancers that include melanomas, basal cell carcinomas, and squamous cell carcinomas. In the United States, the frequency of skin cancers approaches that of all other cancers combined and is on the rise because of the depletion of the ozone layer (10, 21, 23). UV-induced DNA lesions are removed by nucleotide excision repair, but if left unrepaired, they present a block to normal DNA replication. The yeast RAD30 and human RAD30A genes encode a DNA polymerase, polymerase (Pol), which has the unique ability to efficiently and accurately replicate through a UV-induced cis-syn thymine-thymine (TT) dimer, and defects in hRAD30A cause the variant form of xeroderma pigmentosum (12,13,16,22,27). Xeroderma pigmentosum XPV patients suffer from highly elevated levels of sunlight-induced skin cancers.Because of the efficient insertion of As opposite the TT dimer, it has been suggested that Pol is an A rule polymerase (8). In addition to cyclobutane dimers at two adjacent thymines, UV also induces the formation of lesions at dipyrimidine sites that involve a cytosine, most commonly at 5Ј-TC-3Ј and 5Ј-CC-3Ј sequences. In fact, the 3Ј cytosine in both sequence contexts is highly mutagenic, and in both yeast and humans, UV-induced mutations occur primarily by a C3T transition that would result from the insertion of an A opposite the 3Ј damaged C residue during DNA replication (1, 3, 6). If Pol were an A rule polymerase which inserts an A residue by default opposite the various lesions, then the bypass of a CC or a TC cyclobutane dimer by Pol would be mutagenic, not error free as for the TT dimer.In addition to cis-syn cyclobutane pyrimidine dimers, UV induces the formation of pyrimidine (6-4) pyrimidinone photoproducts. The (6-4) photoproduct is formed most frequently at a TC site, whereas the dimer is formed more frequently than the (6-4) photoproduct at a CC site (2, 4, 5). The C of a cis-syn cyclobutane dimer, however, is quite unstable, and in vitro it deaminates to U (24, 25), thus making in vitro bypass studies with TC or CC dimers difficult. Here, we utilize a genetic system designed to test for the role of yeast Pol i...

Mutations in the human CSB gene cause Cockayne syndrome (CS). In addition to increased photosensitivity, CS patients suffer from severe developmental abnormalities, including growth retardation and mental retardation. Whereas a deficiency in the preferential repair of UV lesions from the transcribed strand accounts for the increased photosensitivity of CS patients, the reason for developmental defects in these individuals has remained unclear. Here we provide in vivo evidence for a role of RAD26, the counterpart of the CSB gene in Saccharomyces cerevisiae, in transcription elongation by RNA polymerase II, and in addition we show that under conditions requiring rapid synthesis of new mRNAs, growth is considerably reduced in cells lacking RAD26. These findings implicate a role for CSB in transcription elongation, and they strongly suggest that impaired transcription elongation is the underlying cause of the developmental problems in CS patients.Cockayne syndrome (CS) in humans is characterized by severe growth retardation that has the outward appearance of cachetic dwarfism, and CS patients suffer from progressive neurologic dysfunction and mental retardation. CS individuals also exhibit mild sun sensitivity, but they do not suffer from the increased incidence of skin cancers so prevalent in xeroderma pigmentosum patients. The mean age of death in CS patients is ϳ12 years (13). Mutations in two human genes, CSA and CSB, account for over 90% of CS cases (8). CS cells are impaired in their ability to perform preferential repair of DNA lesions from the transcribed strand (21), a phenomenon known as transcription-coupled repair (TCR) (11). Although the defect in preferential repair of UV lesions from the transcribed strand explains the photosensitivity of CS patients, it fails to account for the characteristic growth and neurological defects associated with CS.RAD26 is the CSB counterpart in Saccharomyces cerevisiae, and inactivation of this gene causes a defect in the TCR of UV-damaged DNA (20). The proteins encoded by the RAD26 and CSB genes are members of the SWI2/SNF2 family of ATPases, and both proteins have DNA-dependent ATPase activities (6, 17). Interestingly, in vitro studies with the purified human CSB protein have suggested a role for CSB as an RNA polymerase II (Pol II) elongation factor (16). Here we utilize S. cerevisiae as a model to investigate the role of RAD26 in transcription elongation in vivo and to examine the possibility that the clinical features of CS patients derive from defects in transcription elongation.Elongation factor SII enables Pol II to transcribe through intrinsic arrest sites in DNA. SII binds arrested Pol II and activates the cleavage of nascent transcript by a latent endoribonuclease intrinsic to Pol II, which eventually results in the clearance of the impediment (15). In S. cerevisiae, DST1, the gene encoding SII, is not essential for viability; however, the dst1⌬ mutant exhibits enhanced sensitivity to the base analog 6-azauracil (6AU) (12), which depletes cellular levels of th...

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Alterations in metabolic pathways are gaining attention as important environmental factors affecting life span, but the determination of specific metabolic pathways and enzymes involved in life span remains largely unexplored. By applying an NMR-based metabolomics approach to a calorie-restricted yeast (Saccharomyces cerevisiae) model, we found that alanine level is inversely correlated with yeast chronological life span. The involvement of the alanine-metabolizing pathway in the life span was tested using a deletion mutant of ALT1, the gene for a key alanine-metabolizing enzyme. The mutant exhibited increased endogenous alanine level and much shorter life span, demonstrating the importance of ALT1 and alanine metabolic pathways in the life span. ALT1's effect on life span was independent of the TOR pathway, as revealed by a tor1 deletion mutant. Further mechanistic studies showed that alt1 deletion suppresses cytochrome c oxidase subunit 2 expression, ultimately generating reactive oxygen species. Overall, ALT1 seems critical in determining yeast life span, and our approach should be useful for the mechanistic studies of life span determinations.