Biomedical subjects

L Prakash

Publications and source records attributed to L Prakash.

At least 73 records · Page 4Linked to original sources

PubMed1996

RAD26, the yeast homolog of human Cockayne's syndrome group B gene, encodes a DNA-dependent ATPase.

Cells from Cockayne's syndrome (CS) patients are sensitive to ultraviolet light and defective in preferential repair of the transcribed DNA strand. CS patients suffer from complex clinical symptoms, including severe growth retardation, neurological degeneration, mental retardation, and cachexia. Two CS complementation groups, CSA and CSB, have been identified so far. RAD26 encodes the yeast counterpart of the CSB gene. Here, we purify Rad26 protein to near homogeneity from yeast cells and show that it is a DNA-dependent ATPase. In contrast to the Mfd protein that functions in transcription-coupled repair in Escherichia coli, and which is a weak and DNA independent ATPase, Rad26 is a much more active ATPase, with a strict dependence on DNA. The possible role of Rad26 ATPase in the displacement of stalled RNA polymerase II from the site of the DNA lesion and in the subsequent recruitment of a DNA repair component is discussed.

Adenosine Triphosphatases↗

PubMed1996

An affinity of human replication protein A for ultraviolet-damaged DNA.

Replication protein A (RPA), a heterotrimeric protein of 70-, 32-, and 14-kDa subunits, is an essential factor for DNA replication. Biochemical studies with human and yeast RPA have indicated that it is a DNA-binding protein that has higher affinity for single-stranded DNA. Interestingly, in vitro nucleotide excision repair studies with purified protein components have shown an absolute requirement for RPA in the incision of UV-damaged DNA. Here we use a mobility shift assay to demonstrate that human RPA binds a UV damaged duplex DNA fragment preferentially. Complex formation between RPA and the UV-irradiated DNA is not affected by prior enzymatic photo-reactivation of the DNA, suggesting an affinity of RPA for the (6-4) photoproduct. We also show that Mg2+ in the millimolar range is required for preferential binding of RPA to damaged DNA. These findings identify a novel property of RPA and implicate RPA in damage recognition during the incision of UV-damaged DNA.

DNA↗

PubMed1996

Reconstitution of TFIIH and requirement of its DNA helicase subunits, Rad3 and Rad25, in the incision step of nucleotide excision repair.

Yeast TFIIH is composed of six subunits: Rad3, Rad25, TFB1, SSL1, p55, and p38. In addition to TFIIH, we have purified a subassembly of the factor that lacks Rad3 and Rad25 and which we refer to as TFIIHi. In the in vitro nucleotide excision repair (NER) system that consists entirely of purified proteins, we show that neither TFIIHi nor a mixture of purified Rad3 and Rad25 proteins is active in NER but that the combination of TFIIHi with Rad3 and Rad25 promotes the incision of UV-damaged DNA. These results provide the first evidence for a direct requirement of Rad3, Rad25, and of one or more of the TFIIHi subunits in the incision step of NER. The NER efficacy of TFIIH is greatly diminished or abolished upon substitution of Rad3 with the rad3 Arg-48 mutant protein or Rad25 with the rad25 Arg-392 mutant protein, respectively, thus indicating a role of the Rad3 and Rad25 DNA helicase functions in the incision of damaged DNA. Our results further indicate that the carboxyl-terminal domain kinase (CTD) TFIIK is dispensable for the incision of damaged DNA in vitro. These studies reveal the differential requirement of Rad3 DNA helicase and CTD kinase activities in damage-specific incision versus RNA polymerase II transcription.

Adenosine Triphosphatases↗

PubMed1996

Nucleotide excision repair in yeast is mediated by sequential assembly of repair factors and not by a pre-assembled repairosome.

In yeast and humans, nucleotide excision repair (NER) of ultraviolet (UV)-damaged DNA requires a large number of highly conserved protein factors, which include the multisubunit RNA polymerase II transcription factor TFIIH. Here, we examine whether NER occurs by sequential assembly of different repair factors at the site of DNA damage or by the placement there of a "preformed" repairosome containing TFIIH and all the other essential NER factors. Contrary to the recent report (Svejstrup, J. Q., Wang, Z., Feaver, W. J., Wu, X., Bushnell, D. A., Donahue, T. F., Friedberg, E. C., and Kornberg, R. D. (1995) Cell 80, 21-28), our results provide no evidence for a pre-assembled repairosome; instead, they support the sequential assembly model. By several independent criteria, including co-purification, immunoprecipitation, and gel filtration of homogeneous proteins, we show that the damage recognition factor Rad14 exists in a ternary complex with the Rad1-Rad10 nuclease. We also find that Rad14 interacts directly with Rad1, but only slightly with Rad10, and that it interacts with the Rad1-Rad10 complex much more efficiently than with Rad1 alone. In the reconstituted NER system, a higher level of incision of UV-damaged DNA is achieved with the Rad1-Rad10-Rad14 complex, which we designate as nucleotide excision repair factor-1, NEF-1.

DNA Damage↗

PubMed1996

Requirement of the yeast MSH3 and MSH6 genes for MSH2-dependent genomic stability.

Defects in DNA mismatch repair result in instability of simple repetitive DNA sequences and elevated levels of spontaneous mutability. The human G/T mismatch binding protein, GTBP/p160, has been suggested to have a role in the repair of base-base and single nucleotide insertion-deletion mismatches. Here we examine the role of the yeast GTBP homolog, MSH6, in mismatch repair. We show that both MSH6 and MSH3 genes are essential for normal genomic stability. Interestingly, although mutations in either MSH3 or MSH6 do not cause the extreme microsatellite instability and spontaneous mutability observed in the msh2 mutant, yeast cells harboring null mutations in both the MSH3 and MSH6 genes exhibit microsatellite instability and mutability similar to that in the msh2 mutant. Results from epistasis analyses indicate that MSH2 functions in mismatch repair in conjunction with MSH3 or MSH6 and that MSH3 and MSH6 constitute alternate pathways of MSH2-dependent mismatch repair.

Amino Acid Sequence↗

PubMed1996

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae.

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3 delta mutation has an effect on recombination similar to that of the rad1 delta and rad10 delta mutations. The msh2 delta mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected.

Amino Acid Sequence↗

PubMed1995

Structure-specific nuclease activity in yeast nucleotide excision repair protein Rad2.

Saccharomyces cerevisiae Rad2 protein functions in the incision step of the nucleotide excision repair of DNA damaged by ultraviolet light. Rad2 was previously shown to act endonucleolytically on circular single-stranded M13 DNA and also to have a 5'-->3' exonuclease activity (Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1993) Nature 366, 365-368; Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1994) J. Biol. Chem. 269, 31342-31345). Using two different branched DNA structures, pseudo Y and flap, we have determined that Rad2 specifically cleaves the 5'-overhanging single strand in these DNAs. Rad2 nuclease is more active on the flap structure than on the pseudo Y structure. Rad2 also acts on a bubble structure that contains an unpaired region of 14 nucleotides, but with a lower efficiency than on the pseudo Y or flap structure. The incision points occur at and around the single strand-duplex junction in the three classes of DNA structures.

Base Sequence↗

PubMed1995

Lethality in yeast of trichothiodystrophy (TTD) mutations in the human xeroderma pigmentosum group D gene. Implications for transcriptional defect in TTD.

Mutations in the human XPD gene result in a defect in nucleotide excision repair of ultraviolet damaged DNA and cause the cancer-prone syndrome xeroderma pigmentosum (XP). Besides XP, mutations in XPD can cause another seemingly unrelated syndrome, trichothiodystrophy (TTD), characterized by sulfur-deficient brittle hair, ichthyosis, and physical and mental retardation. To ascertain the underlying defect responsible for TTD, we have expressed the TTD mutant proteins in the yeast Saccharomyces cerevisiae and determined if these mutations can rescue the inviability of a rad3 null mutation. RAD3, the S. cerevisiae counterpart of XPD, is required for nucleotide excision repair and also has an essential role in RNA polymerase II transcription. Expression of the wild type XPD protein or the XPD Arg-48 protein carrying a mutation in the DNA helicase domain restores viability to the rad3 null mutation. Interestingly, the XPD variants containing TTD mutations fail to complement the lethality of the rad3 null mutation, strongly suggesting that TTD mutations impair the ability of XPD protein to function normally in RNA polymerase II transcription. From our studies, we conclude that XPD DNA helicase activity is not essential for transcription and infer that TTD mutations in XPD result in a defect in transcription.

Amino Acid Sequence↗

PubMed1995

Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA.

Simple repetitive DNA sequences are unstable in human colorectal cancers and a variety of other cancers. Mutations in the DNA mismatch repair genes MSH2, MLH1, and PMS1 result in elevated rates of spontaneous mutation and cause a marked increase in the instability of simple repeats. Compared with the wild type, a null mutation in the yeast RTH1 gene, which encodes a 5' to 3' exonuclease, was shown to increase the rate of instability of simple repetitive DNA by as much as 280 times and to increase the spontaneous mutation rate by 30 times. Epistasis analyses were consistent with the hypothesis that this RTH1-encoded nuclease has a role in the MSH2-MLH-1-PMS1 mismatch repair pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Reconstitution of yeast nucleotide excision repair with purified Rad proteins, replication protein A, and transcription factor TFIIH.

Nucleotide excision repair (NER) functions to remove DNA damage caused by ultraviolet light and by other agents that distort the DNA helix. The NER machinery has been conserved in structure and function from yeast to humans, and in humans, defective NER is the underlying cause of the cancer-prone disease xeroderma pigmentosum. Here, we reconstitute the incision reaction of NER in Saccharomyces cerevisiae using purified protein factors. The Rad14 protein, the Rad4-Rad23 complex, the Rad2 nuclease, the Rad1-Rad10 nuclease, replication protein A, and the RNA polymerase II transcription factor TFIIH were purified to near homogeneity from yeast. We show that these protein factors are both necessary and sufficient for dual incision of DNA damaged by either ultraviolet light or N-acetoxy-2-aminoacetylfluorene. Incision in the reconstituted system requires ATP, which cannot be substituted by adenosine 5'-O-(3-thiotriphosphate), suggesting that the hydrolysis of ATP is indispensable for the incision reaction. The excision DNA fragments formed as a result of dual incision are in the 24-27-nucleotide range.

2-Acetylaminofluorene↗

PubMed1995

Yeast DNA repair protein RAD23 promotes complex formation between transcription factor TFIIH and DNA damage recognition factor RAD14.

In Saccharomyces cerevisiae, the multisubunit RNA polymerase II general transcription factor TFIIH is indispensable for transcription initiation and some of its subunits are known to be required for nucleotide excision repair (NER) of DNA damaged by ultraviolet light. RAD3, a subunit of TFIIH, binds UV-damaged DNA in an ATP-dependent manner. It has, however, remained unclear how TFIIH is assembled with the other damage recognition component RAD14. Here, we demonstrate a higher order complex consisting of TFIIH, RAD14, and another NER protein RAD23, and complex formation between TFIIH and RAD14 is facilitated by the RAD23 protein.

DNA Damage↗

PubMed1995

Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5'- to 3'-exonuclease required for lagging strand DNA synthesis in reconstituted systems.

A 5'- to 3'-exonuclease of about 45 kDa has been purified from various mammalian sources and shown to be required for the completion of lagging strand synthesis in reconstituted DNA replication systems. RTH1 encodes the yeast Saccharomyces cerevisiae counterpart of the mammalian enzyme. To determine the in vivo biological role of RTH1-encoded 5'- to 3'-exonuclease, we have examined the effects of an rth1 delta mutation on various cellular processes. rth1 delta mutants grow poorly at 30 degrees C, and a cessation in growth occurs upon transfer of the mutant to 37 degrees C. At the restrictive temperature, the rth1 delta mutant exhibits a terminal cell cycle morphology similar to that of mutants defective in DNA replication, and levels of spontaneous mitotic recombination are elevated in the rth1 delta mutant even at the permissive temperature. The rth1 delta mutation does not affect UV or gamma-ray sensitivity but enhances sensitivity to the alkylating agent methyl methanesulfonate. The role of RTH1 in DNA replication and in repair of alkylation damage is discussed.

DNA Damage↗

PubMed1995

Mating-type suppression of the DNA-repair defect of the yeast rad6 delta mutation requires the activity of genes in the RAD52 epistasis group.

The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MATa rad6 delta strain can be suppressed by the MAT alpha 2 gene carried on a multicopy plasmid. The a1-alpha 2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-alpha 2 suppression of the rad6 delta mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 delta mutant into the RAD52 recombinational repair pathway. The a1-alpha 2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.

DNA Damage↗

PubMed1994

A conserved 5' to 3' exonuclease activity in the yeast and human nucleotide excision repair proteins RAD2 and XPG.

Saccharomyces cerevisiae RAD2 protein and its human homolog xeroderma pigmentosum group G (XPG) protein function in the incision step of nucleotide excision repair of DNA damaged by ultraviolet light. Both RAD2 and XPG proteins have been shown previously to possess an endonuclease activity. Using DNA substrates labeled at either the 5' end or 3' end, we now demonstrate that RAD2 protein also digests both single-stranded and double-stranded DNAs exonucleolytically with a 5' to 3' directionality. A 5' to 3' exonuclease activity is also present in the XPG protein, indicating evolutionary conservation of this activity. The possible role of RAD2 and XPG 5' to 3' exonuclease activity in nucleotide excision repair is discussed.

Base Sequence↗

PubMed1994

Yeast DNA repair protein RAD5 that promotes instability of simple repetitive sequences is a DNA-dependent ATPase.

The RAD5 gene of Saccharomyces cerevisiae functions in postreplication repair of ultraviolet damaged DNA, and interestingly, it also has a role in increasing the instability of simple repetitive sequences in the genome. In contrast to DNA mismatch repair genes which function in maintaining constant length of repeat sequences, RAD5 promotes alterations in the length of repeat sequences. In this work, we purify the RAD5 protein to near homogeneity from yeast cells and show that it is a single-stranded DNA-dependent ATPase. The possible roles of RAD5 ATPase in postreplication gap filling and in increasing the incidence of length alterations of repeat sequences are discussed.

Adenosine Triphosphatases↗

PubMed1994

Holliday junction cleavage by yeast Rad1 protein.

In Saccharomyces cerevisiae, of the many genes required for excision repair of ultraviolet-damaged DNA, only RAD1 and RAD10 also function in genetic recombination. Complex formation between the RAD1 and RAD10 gene products activates an endonucleolytic function that nicks single-stranded DNA and negatively supercoiled double-stranded DNA. To characterize the recombination role of the proteins Rad1 and Rad10, we have investigated their interaction with the Holliday junction, a four-stranded structure that results from single-stranded crossover between two duplex DNA molecules and whose resolution is obligatory for the generation of mature recombinants. We show that Rad1 binds specifically to a Holliday junction and, in the presence of magnesium, catalyses the endonucleolytic cleavage of the junction. Junction cleavage by Rad1 proceeds sufficiently without Rad10, thus identifying Rad1 as the catalytic subunit of Rad1/Rad10 endonuclease.

Base Sequence↗

PubMed1994

Human xeroderma pigmentosum group G gene encodes a DNA endonuclease.

Because of defective nucleotide excision repair of ultraviolet damaged DNA, xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers. Cell fusion studies have identified seven XP complementation groups, A to G. Previous studies have implicated the products of these seven XP genes in the recognition of ultraviolet-induced DNA damage and in incision of the damage-containing DNA strand. Here, we express the XPG-encoded protein in Sf9 insect cells and purify it to homogeneity. We demonstrate that XPG is a single-strand specific DNA endonuclease, thus identifying the catalytic role of the protein in nucleotide excision repair. We suggest that XPG nuclease acts on the single-stranded region created as a result of the combined action of the XPB helicase and XPD helicase at the DNA damage site.

Animals↗