The chromosomes within eukaryotic cells can be chemically altered through exposure to exogenous chemical and physical DNA damaging agents that are present in the environment [10], [29]. In addition to exogenous sources, DNA is also constantly subject to modification by endogenous cellular processes. Past work has suggested that up to 100,000 DNA lesions are created every day within human cells, caused by a plethora of endogenous chemical and enzymatic processes that are not completely understood [1], [29], [3]. These endogenous processes include hydrolytic deamination and depurination of bases, oxidation caused by reactive oxygen and nitrogen species (RONS) and other radicals, alkylation and formation of other chemical adducts, crosslinking, and many different enzyme-mediated processes. Changes are also produced when structures form in the DNA that interfere with replication such as R-loops, single-strand breaks (SSBs), abasic sites, and other polymerase-stalling lesions [2], [46], [48], [50], [59], [63].

The many types of DNA lesions produced inside cells are repaired by specific repair pathways and repair-related processes. The major DNA repair pathways are mismatch repair (MMR) - fixing wrongly paired DNA bases, nucleotide excision repair (NER) - mainly repairing complex lesions such as ultraviolet light-induced cyclobutane pyrimidine dimers and other intra-strand crosslinks, base excision repair (BER) - replacing damaged or missing bases, plus homologous recombinational repair (HR) and nonhomologous end-joining (NHEJ). These last two pathways are responsible for repairing DNA double-strand breaks (DSBs) [10], [42], [49], [63].

In addition to repair pathways, cells have developed checkpoints that monitor the successful completion of cell cycle events and can initiate a coordinated cellular response when interference is detected. Activation of the DNA damage checkpoint response (DDR) leads to a pause in cell cycling, typically in G1 or G2, which allows extra time for repair to occur before cells proceed into S or M phase [17], [51], [64], [65]. After repair of most lesions occurs, the damage response is reduced and cells resume cycling. Cells of the budding yeast Saccharomyces cerevisiae arrest predominantly in G2 phase after exposure to DNA damaging chemicals or radiation [17], [30], [65]. The arrested cells accumulate in cultures as large-budded, dumbbell-shaped G2 cells that are readily detectable by phase contrast microscopy.

The BER pathway in S. cerevisiae involves the actions of several lesion-specific glycosylases such as Ogg1 (8-oxoguanine DNA N-glycosylase 1), an enzyme that scans DNA for oxidized guanines and excises the damaged bases [28], [52], [9]. In the main line of the pathway, an AP endonuclease activity cleaves a phosphodiester bond on the strand opposite an abasic site to create a single-strand break [6]. DNA polymerase delta may then extend this strand from the 3′ end, leading to displacement of 5–10 nucleotides and creating a flap that is removed by the 5′ flap endonuclease activity of Rad27 (Fen1). Alternatively, AP lyase activities of enzymes such as Ntg1, Ntg2 or Ogg1 may cut opposite the abasic site to produce 3′-blocked ends that are processed by AP endonucleases to create a single nucleotide gap that is filled in by a DNA polymerase. Among the AP endonucleases, Apn1 has the major activity in the cells, accounting for more than 90 % of both AP-endonuclease and 3′-phosphodiesterase activities [6].

apn1 mutants of S. cerevisiae are sensitive to base-damaging agents such as methylmethane sulfonate (MMS) and hydrogen peroxide and exhibit high mutation rates [5], [57], [9]. Unlike apn1 cells, apn2 mutants show normal sensitivity to DNA damaging agents and do not show increased mutation rates. In the absence of both nuclease genes i.e., in apn1 apn2 double mutants, repair of AP sites is severely compromised relative to that seen in the individual single mutants. Mutation frequencies in apn1 apn2 double mutants are much higher than in apn1 or apn2 single mutants [5], [67]. 8-oxoG glycosylase-deficient ogg1 mutants also exhibit reduced survival after exposure to hydrogen peroxide [57].

The HR pathway (also called HDR: homology-directed repair) is the dominant pathway for repair of DSBs within budding yeast cells. After nuclease resection of the ends of a DSB to produce long, RPA protein-coated single-stranded DNA tails, members of the RAD52 group of proteins mediate homology search and strand invasion events that allow the broken DNA to be repaired using an unbroken homologous DNA molecule as a template [39], [47]. Yeast rad52 mutants are extremely sensitive to ionizing radiation and to chemicals capable of producing DSBs either directly or indirectly [37], [47], [53]. In a recent study we observed that rad52 mutants exhibit several additional phenotypes that had not previously been characterized [30]. Cultures of these mutants contain a high percentage of large-budded G2 cells. The fraction of such cells was reduced to wildtype levels when DNA damage checkpoint genes were co-inactivated along with RAD52. These and other experiments indicated that the cells spent extended times in G2 phase due to accumulation of unrepaired damageand persistant activation of the DNA damage checkpoint response. Furthermore, the mutants displayed longer doubling times and larger sizes than wildtype cells and showed striking changes in other morphological and physical properties [30].

In the current study we have expanded our investigation to assess persistent DNA damage stress responses in yeast mutants deficient in each of the five major DNA repair pathways and in the translesion synthesis damage tolerance pathway. Only HR and BER mutants exhibited the high G2 phenotype during normal growth, i.e., in the absence of exogenous DNA damaging agents. Subsequent experiments demonstrated high levels in newly constructed non-library apn1, apn2 and ogg1 mutants using both haploid and diploid cells. The fraction of G2/M cells became synergistically elevated in apn1 apn2 double mutants compared to the single mutants and was also strongly increased when HR and BER mutations were combined. The increase in large-budded cells was eliminated when the RAD9 checkpoint gene was co-inactivated to produce apn1 rad9 or ogg1 rad9 double mutants. Checkpoint-associated and other newly identified phenotypes observed in the BER mutants were strongest in apn1 apn2 double mutants, which have a more complete inactivation of the pathway than any of the BER single mutants. The results demonstrate for the first time that two pathways, HR and BER, are the most critical systems for repair of endogenously produced DNA damage and prevention of transcription and cell cycling stress responses.