Adenovirus ElA transforming function requires two distinct regions of the protein. Transforming activity is closely linked with the presence of a region designated conserved domain 2 and the ability of this region to bind the product of the celular retinoblastoma tumor suppressor gene. We have investigated the biological properties of the second transforming region of ElA, which is located near the N terminus. Transformationdefective mutants containing deletions in the N terminus (deletion of residues between amino acids 2 and 36) were deficient in the ability to induce DNA synthesis and repress insulin enhancer-stimulated activity. The function of the N-terminal region correlated closely with binding of the 300-kilodalton ElA-associated protein and not with binding of the retinoblastoma protein. These results indicate that transformation by ElA is mediated by two functionally independent regions of the protein which interact with different specific cellular proteins and suggest that the 300-kilodalton ElA-associated protein plays a major role in ElA-mediated cell growth control mechanisms.

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation 1 . Defects in crossing over cause infertility, miscarriage and congenital disease. Accordingly, each pair of chromosomes attains at least one crossover through processes that designate and then implement crossing over with high efficiency 2 . At the DNA level, crossing over is implemented through the formation and biased resolution of double-Holliday Junction intermediates [3][4][5] . A central tenet of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands 6 . Although the endonuclease activity of the MutLγ complex has been implicated in crossover-biased resolution [7][8][9][10][11][12] , the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp, PCNA, is important for crossover-biased resolution. In vitro assays with human enzymes show that hPCNA and its loader hRFC are sufficient to activate the hMutLγ endonuclease under physiological conditions.In this context, the hMutLγ endonuclease is further stimulated by a co-dependent activity of the pro-crossover factors hEXO1 and hMutSγ, the latter of which binds Holliday junctions 13 . hMutLγ also specifically binds a variety of branched DNAs, including Holliday junctions, but canonical resolvase activity is not observed implying that the endonuclease incises adjacent to junction branch points to effect resolution. In vivo, we show that budding yeast RFC facilitates MutLγdependent crossing over. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover-resolution and DNA mismatch repair [14][15][16] and evoke a novel model for crossover-specific dHJ resolution during meiosis..

In vivo assembly of plasmids has become an increasingly used process, as high throughput studies in molecular biology seek to examine gene function. In this study, we investigated the plasmid construction technique called gap repair cloning (GRC) in two closely related species of yeast – Saccharomyces cerevisiae and Candida glabrata. GRC utilizes homologous recombination (HR) activity to join a linear vector and a linear piece of DNA that contains base pair homology. We demonstrate that a minimum of 20 bp of homology on each side of the linear DNA is required for GRC to occur with at least 10% efficiency. Between the two species, we determine that S. cerevisiae is slightly more efficient at performing GRC. GRC is less efficient in rad52 deletion mutants, which are defective in HR in both species. In dnl4 deletion mutants, which perform less non-homologous end joining (NHEJ), the frequency of GRC increases in C. glabrata, whereas GRC frequency only minimally increases in S. cerevisiae, suggesting that NHEJ is more prevalent in C. glabrata. Our studies allow for a model of the fate of linear DNA when transformed into yeast cells. This model is not the same for both species. Most significantly, during GRC, C. glabrata performs NHEJ activity at a detectable rate (>5%), while S. cerevisiae does not. Our model suggests that S. cerevisiae is more efficient at HR because NHEJ is less prevalent than in C. glabrata. This work demonstrates the determinants for GRC and that while C. glabrata has a lower efficiency of GRC, this species still provides a viable option for GRC.

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation 1 . Defects in crossing over cause infertility, miscarriage and congenital disease. Accordingly, each pair of chromosomes attains at least one crossover through processes that designate and then implement crossing over with high efficiency 2 . At the DNA level, crossing over is implemented through the formation and biased resolution of double-Holliday Junction intermediates [3][4][5] . A central tenet of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands 6 . Although the endonuclease activity of the MutLγ complex has been implicated in crossover-biased resolution 7-12 , the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp, PCNA, is important for crossover-biased resolution. In vitro assays with human enzymes show that hPCNA and its loader hRFC are sufficient to activate the hMutLγ endonuclease under physiological conditions. In this context, the hMutLγ endonuclease is further stimulated by a co-dependent activity of the pro-crossover factors hEXO1 and hMutSγ, the latter of which binds Holliday junctions 13 . hMutLγ also specifically binds a variety of branched DNAs, including Holliday junctions, but canonical resolvase activity is not observed implying that the endonuclease incises adjacent to junction branch points to effect resolution. In vivo, we show that budding yeast RFC facilitates MutLγdependent crossing over. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover-resolution and DNA mismatch repair [14][15][16] and evoke a novel model for crossover-specific dHJ resolution during meiosis. MainMeiotic recombination is initiated by programmed DNA double-strand breaks (DSBs) and proceeds via homologous pairing and DNA strand-exchange to form joint-molecule (JM) intermediates 1 . Regulatory processes designate a subset of events to become crossovers, and at these sites nascent JMs are matured into double-Holliday junctions (dHJs). Through poorly defined mechanisms, MutLγ (comprising MLH1 and MLH3) accumulates at prospective crossover sites and biases dHJ resolution to specifically produce crossovers 17,18 . When MutLγ is dysfunctional, dHJs are still resolved, but with a non-crossover outcome 8 . Consequently, chromosome segregation fails and fertility is diminished. MLH1 and MLH3 are conserved members of the MutL family of DNA mismatch-repair (MMR) factors that couple mismatchrecognition by a MutS complex to downstream excision and resynthesis of the nascent strand 14,16 . MutL complexes from diverse species possess endonuclease activity that provides an initiation site for mismatch excision by a 5'-3 exonuclease such as EXO1 15 . During DNA replication, MutL-catalyzed incision, and thus MMR, is specifically targeted to the nascent strand via an orientation-specific...