Yong Hwan Jin

Identification of the DNA damage-responsive elements of therhp51+ gene, arecA andRAD51 homolog from the fission yeastSchizosaccharomyces pombe

TheSchizosaccharomyces pombe rhp51+ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and theSaccharomyces cerevisiae RAD51 protein. Levels ofrhp51+ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. Anrhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulatingrhp51+ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (fordamage-responsiveelement), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes fromS. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility ofrhp51+ expression.

Isolation and characterization of hrp1+, a new member of the SNF2/SWI2 gene family from the fission yeast Schizosaccharomyces pombe

Abstract The SNF2/SWI2 ATPase/helicase family comprises proteins from a variety of species, which serve a number of functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair. Several proteins with unknown functions are also included in this family. The number of genes that belong to this family is rapidly expanding, which makes it easier to analyze the common biological functions of the family members. This study was designed to clone the SNF2/SWI2 helicase-related genes from the fission yeast Schizosaccharomyces pombe in the hope that this would help to elucidate the common functions of the proteins in this family. The hrp1+ (helicase-related gene from S. pombe) gene was initially cloned by PCR amplification using degenerate primers based on conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair. The hrp1+ ORF codes for an 1373-amino acid polypeptide with a molecular mass of 159 kDa. Like other SNF2/SWI2 family proteins, the deduced amino acid sequence of Hrp1 contains DNA-dependent ATPase/7 helicase domains, as well as a chromodomain and a DNA-binding domain. This configuration is similar to that of mCHD1 (mouse chromo-ATPase/helicase-DNA-binding protein 1), suggesting that Hrp1 is a S. pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to facilitate gene expression. Northern blot analysis showed that the hrp1+ gene produces a 4.6-kb transcript, which reaches its maximal level just before the cells enter the exponential growth phase, and then decreases gradually. DNA-damaging agents, such as MMS, MNNG and UV, decrease the rate of transcription of hrp1+. Deletion of the hrp1+ gene resulted in accelerated cell growth. On the other hand, overexpression of Hrp1 caused a reduction in growth rate. These results indicate that hrp1+ may act as a negative regulator of cellular growth.

Identification and expression of uvi31(+), a UV-inducible gene from Schizosaccharomyces pombe

The Schizosaccharomyces pombe uvi31+ gene has been previously isolated as a UV‐inducible gene [Lee JK et al. (1994) Biochem Biophys Res Commun 202:1113–1119]. This gene encodes a protein of about 12 kDa with 57% amino acid sequence similarity to Escherichia coli BolA protein which is known to be involved in switching between the cell elongation and septation systems during the cell division cycle. The putative Mlul cell cycle box (MCB), SWI4/6‐dependent cell cycle box (SCB), and gear‐box elements are found in the upstream region of uvi31+ gene, suggesting that this gene shows the cell cycle‐regulated and growth phase‐dependent expression. Interestingly, the level of uvi31− transcript varies throughout the cell cycle, peaking in G1 phase before septation, and also shows the growth phase‐dependent pattern during cellular growth, increasing maximally at the diauxic shift phase just before stationary phase. Furthermore, the transcript level of this gene is raised after S phase arrest, and is also increased maximally at 4 hr after UV irradiation of 240 J/m2. These results suggest that the delayed induction of uvi31+ gene after UV irradiation may be caused by cell cycle control of this gene after DNA replication checkpoint arrest. Thus, the uvi31+ gene may play a role in controlling the progress of the cell cycle after DNA damage (UV irradiation). Environ. Mol. Mutagen. 30:72–81, 1997

Purification and Characterization of Hrp1, a Homolog of Mouse CHD1 from the Fission Yeast Schizosaccharomyces pombe

Hrp1, of Schizosaccharomyces pombe, is a new member of the SWI2/SNF2 protein family that contains a chromodomain and a DNA binding domain as well as ATPase/7 helicase domains. This configuration suggests that Hrp1 could be a homolog of mouse CHD1, which is thought to function in altering the chromatin structure to facilitate gene expression. To understand the enzymatic nature of Hrp1, we purified the 6‐Histidine‐tagged Hrp1 protein (6×His‐Hrp1) to homogeneity from a S. pombe Hrp1‐overexpressing strain and then examined its biochemical properties. We demonstrate that the purified 6×His‐Hrp1 protein exhibited a DNA‐binding activity with a moderate preference to the (A+T)‐rich tract in double‐stranded DNA via a minor groove interaction. However, we failed to detect any intrinsic DNA helicase activity from the purified Hrp1 like other SWI2/SNF2 proteins. These observations suggest that the DNA binding activities of Hrp1 may be involved in the remodeling of the chromatin structure with DNA‐dependent ATPase. We...