Homologous recombination (HR) is a ubiquitous cellular pathway that mediates transfer of genetic information between homologous or near homologous (homeologous) DNA sequences. During meiosis it ensures proper chromosome segregation in the first division. Moreover, HR is critical for the tolerance and repair of DNA damage, as well as in the recovery of stalled and broken replication forks. Together these functions preserve genomic stability and assure high fidelity transmission of the genetic material in the mitotic and meiotic cell divisions. This review will focus on the Rad54 protein, a member of the Snf2-family of SF2 helicases, which translocates on dsDNA but does not display strand displacement activity typical for a helicase. A wealth of genetic, cytological, biochemical and structural data suggests that Rad54 is a core factor of HR, possibly acting at multiple stages during HR in concert with the central homologous pairing protein Rad51.

The mechanism by which trinucleotide expansion occurs in human genes is not understood. However, it has been hypothesized that DNA secondary structure may actively participate by preventing FEN-1 cleavage of displaced Okazaki fragments. We show here that secondary structure can, indeed, play a role in expansion by a FEN-1-dependent mechanism. Secondary structure inhibits flap processing at CAG, CGG, or CTG repeats in a length-dependent manner by concealing the 5' end of the flap that is necessary for both binding and cleavage by FEN-1. Thus, secondary structure can defeat the protective function of FEN-1, leading to site-specific expansions. However, when FEN-1 is absent from the cell, alternative pathways to simple inhibition of flap processing contribute to expansion.

An ␣-amylase was purified from culture supernatants of Sulfolobus solfataricus 98/2 during growth on starch as the sole carbon and energy source. The enzyme is a homodimer with a subunit mass of 120 kDa. It catalyzes the hydrolysis of starch, dextrin, and ␣-cyclodextrin with similar efficiencies. Addition of exogenous glucose represses production of ␣-amylase, demonstrating that a classical glucose effect is operative in this organism. Synthesis of [35 S]-␣-amylase protein is also subject to the glucose effect. ␣-Amylase is constitutively produced at low levels but can be induced further by starch addition. The absolute levels of ␣-amylase detected in culture supernatants varied greatly with the type of sole carbon source used to support growth. Aspartate was identified as the most repressing sole carbon source for ␣-amylase production, while glutamate was the most derepressing. The pattern of regulation of ␣-amylase production seen in this organism indicates that a catabolite repression-like system is present in a member of the archaea.Catabolite repression is a paradigm for studies concerned with global and specific gene control mechanisms (22). In prokaryotes, catabolite repression together with transient repression and inducer exclusion make up what has been termed the glucose effect or repression of catabolic enzyme synthesis by glucose (23). However, for eukaryotes, the term catabolite repression is more generally used as a pseudonym for the glucose effect. In fact, evidence for transient repression, inducer exclusion, and requisite aspects of catabolite repression, including the ability to grow most rapidly on preferred carbon sources, is not well demonstrated (for reviews, see references 29 and 31). Catabolite repression in prokaryotes and eukaryotes has received wide attention, but the existence of an analogous process in the archaea has not been addressed. One hallmark of this process in gram-negative bacteria consists of the global mode of gene regulation of catabolite-repressible genes mediated by the small molecule cyclic AMP (cAMP) (8). Although the role of cAMP in some prokaryotes is well accepted, it has been eliminated as an effector in the corresponding catabolite response in the gram-positive bacterium Bacillus subtilis (for a review, see reference 14). In eukaryotes, including the budding yeast Saccharomyces cerevisiae, cAMP plays a crucial but indirect role in mediating the glucose effect. Interestingly, cAMP has been reported in a range of archaea (21).Sulfolobus solfataricus is an extremely thermophilic organism which inhabits acidic hot springs. S. solfataricus is a member of the archaea and has been assigned to a subdivision termed the crenarchaeota by rRNA gene (rDNA) sequence analysis (32). It is capable of diverse modes of metabolism at temperatures ranging between 70 and 90ЊC. It can grow either lithoautotrophically, oxidizing sulfur (4, 15), or chemoheterotrophically on starch or other sugars as sole carbon and energy sources (7,11). Recent studies also suggest that hot springs cont...

A soluble maltase (␣-glucosidase) with an apparent subunit mass of 80 kDa was purified to homogeneity from Sulfolobus solfataricus. The enzyme liberates glucose from maltose and malto-oligomers. Maximal activity was observed at 105؇C, with half-lives of 11 h (85؇C), 3.0 h (95؇C), and 2.75 h (100؇C). The enzyme was generally resistant to proteolysis and denaturants including aliphatic alcohols. n-Propanol treatment at 85؇C increased both K m and V max for maltose hydrolysis.Sulfolobus solfataricus (5) is an aerobic, extremely thermophilic microorganism found in acidic hotsprings. It has been assigned to the subgroup Crenarchaeota within the domain Archaea by 16S rDNA sequence analysis (17). S. solfataricus has the ability to grow lithoautotrophically utilizing sulfur as an energy source or chemoheterotrophically on a variety of sugars and polysaccharides, including starch, as sole carbon and energy sources (2, 5, 7). Starch catabolism frequently depends upon a secreted ␣-amylase which generates linear maltodextrins from starch as well as a cell-associated ␣-glucosidase (maltase) which converts maltose and maltodextrins to glucose (11). Extremely thermophilic ␣-amylases have been identified in members of the obligately anaerobic sulfur-reducing genus Pyrococcus (3, 13, 15), which is classified within the euryarchaeotal branch of the domain Archaea. A monomeric 125-kDa ␣-glucosidase has also been identified in this group (4, 12). Several lines of evidence suggested that an ␣-glucosidase was similarly present in the crenarchaeote S. solfactaricus. These included the ability of the organism to utilize maltose as the sole carbon and energy source (7) and the presence of a p-nitrophenyl-␣-D-glucopyranoside (PNPG) hydrolytic activity in crude cell extracts (1).S. solfataricus 98/2. S. solfataricus 98/2 was obtained from L. Hochstein (10). Cells were cultured at 80ЊC as described previously (2) at pH 3.0 with maltose at 0.2% (wt/vol) as the sole carbon and energy source. Cultures were grown in 500-ml volumes and were harvested in late exponential phase, which was equivalent to a cell density of 10 9 cells per ml and an A 540 of 1.0. Cells were recovered by centrifugation at 4ЊC, and the resulting cell pellet was frozen at Ϫ20ЊC. Species identification was determined by rDNA sequence analysis (9). Such information was important in light of the confusion regarding Sulfolobus isolates (14,18). DNA sequence analysis of a 487-bp 16S rDNA sequence indicated that strain 98/2 shared 100% homology with S. solfataricus P2, 98.8% homology with S. shibatae B12 (6), and 87.6% homology with S. acidocaldarius (9,14,18). In addition, S. solfataricus P2 and 98/2 utilize a range of sugars, including maltose, mannose, and trehalose, which cannot be utilized by S. acidocaldarius, as well as cellobiose, which is used either poorly or not at all by either S. acidocaldarius or S. shibatae (7).Purification of the S. solfataricus maltase. The colorimetric maltose analog PNPG was used as a substrate to monitor maltase activity during the purifica...

DNA damage checkpoints coordinate the cellular response to genotoxic stress (10,45,52,82). In the budding yeast Saccharomyces cerevisiae, the DNA damage checkpoints are largely controlled by the phosphatidyl-inositol 3-kinase-like kinase Mec1, an ortholog of the human ATM and ATR kinases. Via the Rad9 and Mrc1 adaptor proteins, Mec1 controls the downstream kinases Chk1 and Rad53. This process amplifies the checkpoint response and transforms localized Mec1 activation into a pan-nuclear response regulating downstream effector pathways, including cell cycle control, transcription, DNA replication, and possibly DNA damage repair and DNA damage tolerance pathways.Checkpoint mutants fail to arrest their cell cycles in response to DNA damage and replication fork stalling, leading to damage sensitivity and genomic instability (49). However, extrinsically imposed cell cycle arrest does not rescue the damage sensitivity of S. cerevisiae rad53 or mec1 mutants (4, 65) or human ATM-deficient cells (73; reviewed in reference 24) and only partially rescues the sensitivity of S. cerevisiae rad9 cells (74), suggesting that DNA damage checkpoints also regulate mechanisms other than cell cycle arrest that are critical for survival and genome stability.Stalled replication forks are considered a major source of genomic instability (29), and multiple pathways operate at stalled forks, presumably in a hierarchy that is under active regulation. An analysis of a mec1 hypomorphic mutant demonstrated a central role of DNA damage checkpoints in preventing irreversible breakdown of stalled replication forks in budding yeast (66). The postreplication repair (PRR) controlled by the Rad6-Rad18 proteins is critical to budding yeast for the toleration of replication-blocking lesions (8). PRR comprises a number of pathways, which are incompletely understood at this moment, involving translesion synthesis (TLS) by DNA polymerases and template switching. TLS polymerases, including REV3, which encodes a subunit of DNA polymerase zeta (Pol), and RAD30, which encodes Pol in S. cerevisiae, accommodate damaged DNA templates, leading to bypass and damage tolerance. Template switching can occur by fork regression, a process that appears to be controlled by the Rad5 protein. However, the subpathways in PRR are complex and roles of Rad5 in conjunction with the TLS polymerase Rev3 have been identified (13,47). Template switching can also be catalyzed during gap repair by homologous recombination (HR) mediated by the RAD52 epistasis group (31).HR is a major pathway for the repair of DNA doublestranded breaks (DSBs) and other types of DNA damage. In bacteria, recombination is central in the recovery of stalled