The SNF2 family of proteins includes representatives from a variety of species with roles in cellular processes such as transcriptional regulation (e.g. MOT1, SNF2 and BRM), maintenance of chromosome stability during mitosis (e.g. lodestar) and various aspects of processing of DNA damage, including nucleotide excision repair (e.g. RAD16 and ERCC6), recombinational pathways (e.g. RAD54) and post-replication daughter strand gap repair (e.g. RAD5). This family also includes many proteins with no known function. To better characterize this family of proteins we have used molecular phylogenetic techniques to infer evolutionary relationships among the family members. We have divided the SNF2 family into multiple subfamilies, each of which represents what we propose to be a functionally and evolutionarily distinct group. We have then used the subfamily structure to predict the functions of some of the uncharacterized proteins in the SNF2 family. We discuss possible implications of this evolutionary analysis on the general properties and evolution of the SNF2 family.

DNAs that contain specific yeast chromosomal sequences called ARSs transform Saccharomyces cerevisiae at high frequency and can replicate extrachromosomally as plasmids when introduced into S. cerevisiae by transformation. To determine the boundaries of the minimal sequences required for autonomous replication in S. cerevisiae, we have carried out in vitro mutagenesis of the first chromosomal ARS described, ARSI. Rather than identifying a distinct and continuous segment that mediates the ARS+ phenotype, we find three different functional domains within ARS1. We define domain A as the 11-base-pair (bp) sequence that is also found at most other ARS regiQns. It is necessary but not sufficient for high-frequency transformation. Domain B, which cannot mediate high-frequency transformation, or replicate by itself, is required for efficient, stable replication of plasmids containing domain A. Domain B, as we define it, is continuous with domain A in ARSI, but insertions of 4 bp between the two do not affect replication. The extent of domain B has an upper limit of 109 bp and a lower limit of 46 bp in size. There is no obvious sequence homology between domain B of ARS) and any other ARS sequence. Finally, domain C is defined on the basis of our deletions as at least 200 bp flanking domain A on the opposite side from domain B and is also required for the stability of domain A in S. cerevisiae. The effect of deletions of domain C can be observed only in the absence of domain B, at least by the assays used in the current study, and the significance of this finding is discussed.As in higher organisms, chromosome replication in Saccharomyces cerevisiae initiates at multiple sites and is bidirectional. The DNA is replicated only during the S phase, and activation of individual replicons is thought to occur according to a specific temporal program, with each replicon activated only once per cell cycle (for review, see reference 3). There is no direct evidence in any eucaryote that chromosomal replication initiates at the same chromosomal sequence during each cell cycle. However, indirect evidence is available in S. cerevisiae. Yeast sequences have been isolated that confer on any colinear DNA the ability to be maintained in S. cerevisiae as autonomously replicating, albeit highly unstable, plasmids (20,41,44). The sequences responsible for autonomous replication are designated ARS.Estimates of the number of ARS elements in the S. cerevisiae genome (1, 7) agree with the number of initiation sites for DNA replication estimated by fiber autoradiography and electron microscopy (reviewed in reference 3). ARS elenlents have been classified by nucleic acid hybridization as either unique or repetitive (6,8,41,45

While preferential repair of the transcribed strands within active genes has been demonstrated in organisms as diverse as humans and Escherichia colt, it has not previously been shown to occur in chromosomal genes in the yeast Saccharomyces cerevisiae. We found that repair of cyclobutane pyrimidine dimers in the transcribed strand of the expressed RPB2 gene in the chromosome of a repair-proficient strain is much more rapid than that in the nontranscribed strand. Furthermore, a copy of the RPB2 gene borne on a centromeric ARSI plasmid showed the same strand bias in repair. To investigate the relation of this strand bias to transcription, we studied repair in a yeast strain with the temperature-sensitive mutation, rpbl-1, in the largest subunit of RNA polymerase II. When exponentially growing rpbl-1 cells are shifted to the nonpermissive temperature, they rapidly cease mRNA synthesis. At the permissive temperature, both rpbl-1 and the wild-type, parental cells exhibited rapid, proficient repair in the transcribed strand of chromosomal and plasmid-borne copies of the RPB2 gene. At the nonpermissive temperature, the rate of repair in the transcribed strand in rpbl-1 cells was reduced to that in the nontranscribed strand.These findings establish the dependence of strand bias in repair on transcription by RNA polymerase II in the chromosomes and in plasmids, and they validate the use of plasmids for analysis of the relation of repair to transcription in yeast.Both eukaryotes and prokaryotes carry out excision repair of DNA damage after exposure to UV light (1). The two major classes of lesions produced are 5-5, 6-6 cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts, both of which are removed from the DNA. Furthermore, CPD removal has been shown in mammalian cells to be faster in transcriptionally active genes when compared to the genome overall (for recent reviews, see refs. 2 and 3) and more rapid in the transcribed strand in expressed genes than in the nontranscribed strand (4). This preferential repair of the transcribed strand in expressed genes has also been shown for the lac genes in the Escherichia coli chromosome (5) and for the uvrC gene in vitro (6). Thus, it seems likely that preferential DNA repair of active genes is a conserved pathway for nucleotide excision repair in both prokaryotes and eukaryotes. However, preferential DNA repair of the transcribed strand of an active chromosomal gene has not yet been shown in the unicellular eukaryote Saccharomyces cerevisiae.Recent reports of transcription-associated nucleotide excision repair in yeast are seemingly inconsistent. Studies of repair in unique chromosomal DNA sequences did not show a strand bias in repair (7-9). In contrast, Smerdon and Thoma and colleague (10,11) reported that excision repair of CPDs in a plasmid was rapid for transcribed strands, while nontranscribed sequences were slowly repaired. To resolve these differences we have compared the repair rates in the same expressed sequence in a plasmid...

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The gene encoding a yeast ARS-binding protein, ABF I, has been cloned by screening a genomic Xgtll library using monoclonal and polyclonal antibodies against ABF I. ABF I is of interest because it not only binds to ARSs but also to the 5'-flanking region of genes encoding proteins involved in transcription, translation, respiration, and cell-cycle control. The cloned gene has been used to prepare null mutants, which further demonstrate the importance of the ABF I protein by showing that it is essential for vegetative growth. ABFl maps to chromosome V. The DNA sequence of the ABFl gene reveals several motifs characteristic of DNAbinding proteins but shows no overall similarity to any protein of known function.