PubMed Health⌕ Search

Biomedical subjects

L Prakash

Publications and source records attributed to L Prakash.

At least 37 records · Page 2Linked to original sources
PubMed2000

Mismatch extension ability of yeast and human DNA polymerase eta.

DNA polymerase eta (Poleta) functions in error-free replication of UV-damaged DNA, and in vitro it efficiently bypasses a cis-syn T-T dimer by incorporating two adenines opposite the lesion. Steady state kinetic studies have shown that both yeast and human Poleta are low-fidelity enzymes, and they misincorporate nucleotides with a frequency of 10(-2)-10(-3) on both undamaged and T-T dimer-containing DNA templates. To better understand the role of Poleta in error-free translesion DNA synthesis, here we examine the ability of Poleta to extend from base mismatches. We find that both yeast and human Poleta extend from mismatched base pairs with a frequency of approximately 10(-3) relative to matched base pairs. In the absence of efficient extension of mismatched primer termini, the ensuing dissociation of Poleta from DNA may favor the excision of mismatched nucleotides by a proofreading exonuclease. Thus, we expect DNA synthesis by Poleta to be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

Base Pair Mismatch↗
PubMed2000

Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions.

DNA lesions can often block DNA replication, so cells possess specialized low-fidelity, and often error-prone, DNA polymerases that can bypass such lesions and promote replication of damaged DNA. The Saccharomyces cerevisiae RAD30 and human hRAD30A encode Pol eta, which bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Here we show that a related human gene, hRAD30B, encodes the DNA polymerase Pol iota, which misincorporates deoxynucleotides at a high rate. To bypass damage, Pol iota specifically incorporates deoxynucleotides opposite highly distorting or non-instructional DNA lesions. This action is combined with that of DNA polymerase Pol zeta, which is essential for damage-induced mutagenesis, to complete the lesion bypass. Pol zeta is very inefficient in inserting deoxynucleotides opposite DNA lesions, but readily extends from such deoxynucleotides once they have been inserted. Thus, in a new model for mutagenic bypass of DNA lesions in eukaryotes, the two DNA polymerases act sequentially: Pol iota incorporates deoxynucleotides opposite DNA lesions, and Pol zeta functions as a mispair extender.

Base Pairing↗
PubMed2000

Apurinic endonuclease activity of yeast Apn2 protein.

Abasic (apurinic/apyrimidinic; AP) sites are generated in vivo through spontaneous base loss and by enzymatic removal of bases damaged by alkylating agents and reactive oxygen species. In Saccharomyces cerevisiae, the APN1 and APN2 genes function in alternate pathways of AP site removal. Apn2-like proteins have been identified in other eukaryotes including humans, and these proteins form a distinct subfamily within the exonuclease III (ExoIII)/Ape1/Apn2 family of proteins. Apn2 and other members of this subfamily contain a carboxyl-terminal extension not present in the ExoIII/Ape1-like proteins. Here, we purify the Apn2 protein from yeast and show that it is a class II AP endonuclease. Deletion of the carboxyl terminus does not affect the AP endonuclease activity of the protein, but this protein is defective in the removal of AP sites in vivo. The carboxyl terminus may enable Apn2 to complex with other proteins, and such a multiprotein assembly may be necessary for the efficient recognition and cleavage of AP sites in vivo.

Amino Acid Sequence↗
PubMed2000

Nucleotide excision repair in yeast.

In nucleotide excision repair (NER) in eukaryotes, DNA is incised on both sides of the lesion, resulting in the removal of a fragment approximately 25-30 nucleotides long. This is followed by repair synthesis and ligation. The proteins encoded by the various yeast NER genes have been purified, and the incision reaction reconstituted in vitro. This reaction requires the damage binding factors Rad14, RPA, and the Rad4-Rad23 complex, the transcription factor TFIIH which contains the two DNA helicases Rad3 and Rad25, essential for creating a bubble structure, and the two endonucleases, the Rad1-Rad10 complex and Rad2, which incise the damaged DNA strand on the 5'- and 3'-side of the lesion, respectively. Addition of the Rad7-Rad16 complex to this reconstituted system stimulates the incision reaction many fold. The various NER proteins exist in vivo as part of multiprotein subassemblies which have been named NEFs (nucleotide excision repair factors). Rad14 and Rad1-Rad10 form one subassembly called NEF1, the Rad4-Rad23 complex is named NEF2, Rad2 and TFIIH constitute NEF3, and the Rad7-Rad16 complex is called NEF4. Although much has been learned from yeast about the function of NER genes and proteins in eukaryotes, the underlying mechanisms by which damage is recognized, NEFs are assembled at the damage site, and the DNA is unwound and incised, remain to be elucidated.

Adenosine Triphosphatases↗
PubMed2000

The human DINB1 gene encodes the DNA polymerase Poltheta.

The human DINB1 gene shares a high degree of homology with the Escherichia coli dinB gene. Here, we purify the hDINB1-encoded protein and show that it is a DNA polymerase. Because hDinB1 is the eighth eukaryotic DNA polymerase to be described, we have named it DNA polymerase (Pol) theta. hPoltheta is unable to bypass a cis-syn thymine-thymine dimer, nor does it bypass a (6-4) photoproduct or an abasic site. We also examine the fidelity of hPoltheta on nondamaged DNA templates by steady-state kinetic analyses and find that hPoltheta misincorporates deoxynucleotides with a frequency of about 10(-3) to 10(-4). We discuss the relationship between the fidelity of hPoltheta and its inability to bypass DNA damage.

Base Sequence↗
PubMed2000

Accuracy of thymine-thymine dimer bypass by Saccharomyces cerevisiae DNA polymerase eta.

The Saccharomyces cerevisiae RAD30 gene functions in error-free replication of UV-damaged DNA. RAD30 encodes a DNA polymerase, Pol eta, which inserts two adenines opposite the two thymines of a cis-syn thymine-thymine (T-T) dimer. Here we use steady-state kinetics to determine the accuracy of DNA synthesis opposite the T-T dimer. Surprisingly, the accuracy of DNA synthesis opposite the damaged DNA is nearly indistinguishable from that opposite nondamaged DNA, with frequencies of misincorporation of about 10(-2) to 10(-3). These studies support the hypothesis that unlike most DNA polymerases, Pol eta is able to tolerate distortions in DNA resulting from damage, which then enables the polymerase to utilize the intrinsic base pairing ability of the T-T dimer.

Base Sequence↗
PubMed2000

Fidelity of human DNA polymerase eta.

Xeroderma pigmentosum (XP) patients are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers. The variant form of XP results from mutations in the hRAD30A gene, which encodes the DNA polymerase in humans, hPol(eta). Of the eukaryotic DNA polymerases, only human Pol(eta) and its yeast counterpart have the ability to replicate DNA containing a cis-syn thymine-thymine (T-T) dimer. Here we measure the fidelity of hPol(eta) on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer. Opposite all four nondamaged template bases, hPol(eta) misincorporates nucleotides with a frequency of approximately 10(-2)-10(-3), and importantly, hPol(eta) synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA. The low fidelity of hPol(eta) may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer.

DNA Damage↗
PubMed2000

Piperine derived from black pepper increases the plasma levels of coenzyme Q10 following oral supplementation.

An extract from the fruits of black pepper consisting of a minimum of 98% pure piperine was evaluated in a clinical study using a double-blind design. The relative bioavailability of 90 mg and 120 mg of coenzyme Q10 administered in a single-dose experiment or in separate experiments for 14 and 21 days with placebo or with 5 mg of piperine was determined by comparing measured changes in plasma concentration. The inter-subject variability was minimized by limiting the selection of individuals to healthy adult male volunteers with (presupplementation) fasting coenzyme Q10 values between 0.30 and 0.60 mg/L. The results of the single-dose study and the 14-day study indicate smaller, but not significant, increases in plasma concentrations of coenzyme Q10 in the control group compared with the group receiving coenzyme Q10 with a supplement of piperine. Supplementation of 120 mg coenzyme Q10 with piperine for 21 days produced a statistically significant (p = 0.0348), approximately 30% greater, area under the plasma curve than was observed during supplementation with coenzyme Q10 plus placebo. It is postulated that the bioenhancing mechanism of piperine to increase plasma levels of supplemental coenzyme Q10 is nonspecific and possibly based on its description in the literature as a thermonutrient.

Journal Article↗
PubMed2000

Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase eta.

Oxidative damage to DNA has been proposed to have a role in cancer and ageing. Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA, and 7, 8-dihydro-8-oxoguanine (8-oxoG) is one of the adducts formed. Eukaryotic replicative DNA polymerases replicate DNA containing 8-oxoG by inserting an adenine opposite the lesion; consequently, 8-oxoG is highly mutagenic and causes G:C to T:A transversions. Genetic studies in yeast have indicated a role for mismatch repair in minimizing the incidence of these mutations. In Saccharomyces cerevisiae, deletion of OGG1, encoding a DNA glycosylase that functions in the removal of 8-oxoG when paired with C, causes an increase in the rate of G:C to T:A transversions. The ogg1Delta msh2Delta double mutant displays a higher rate of CAN1S to can1r forward mutations than the ogg1Delta or msh2Delta single mutants, and this enhanced mutagenesis is primarily due to G:C to T:A transversions. The gene RAD30 of S. cerevisiae encodes a DNA polymerase, Poleta, that efficiently replicates DNA containing a cis-syn thymine-thymine (T-T) dimer by inserting two adenines across from the dimer. In humans, mutations in the yeast RAD30 counterpart, POLH, cause the variant form of xeroderma pigmentosum (XP-V), and XP-V individuals suffer from a high incidence of sunlight-induced skin cancers. Here we show that yeast and human POLeta replicate DNA containing 8-oxoG efficiently and accurately by inserting a cytosine across from the lesion and by proficiently extending from this base pair. Consistent with these biochemical studies, a synergistic increase in the rate of spontaneous mutations occurs in the absence of POLeta in the yeast ogg1Delta mutant. Our results suggest an additional role for Poleta in the prevention of internal cancers in humans that would otherwise result from the mutagenic replication of 8-oxoG in DNA.

DNA↗
PubMed2000

Evidence for the involvement of nucleotide excision repair in the removal of abasic sites in yeast.

In eukaryotes, DNA damage induced by ultraviolet light and other agents which distort the helix is removed by nucleotide excision repair (NER) in a fragment approximately 25 to 30 nucleotides long. In humans, a deficiency in NER causes xeroderma pigmentosum (XP), characterized by extreme sensitivity to sunlight and a high incidence of skin cancers. Abasic (AP) sites are formed in DNA as a result of spontaneous base loss and from the action of DNA glycosylases involved in base excision repair. In Saccharomyces cerevisiae, AP sites are removed via the action of two class II AP endonucleases, Apn1 and Apn2. Here, we provide evidence for the involvement of NER in the removal of AP sites and show that NER competes with Apn1 and Apn2 in this repair process. Inactivation of NER in the apn1Delta or apn1Delta apn2Delta strain enhances sensitivity to the monofunctional alkylating agent methyl methanesulfonate and leads to further impairment in the cellular ability to remove AP sites. A deficiency in the repair of AP sites may contribute to the internal cancers and progressive neurodegeneration that occur in XP patients.

Alkylating Agents↗
PubMed2000

Replication past O(6)-methylguanine by yeast and human DNA polymerase eta.

O(6)-Methylguanine (m6G) is formed by the action of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on DNA. m6G is a highly mutagenic and carcinogenic lesion, and it presents a block to synthesis by DNA polymerases. Here, we provide genetic and biochemical evidence for the involvement of yeast and human DNA polymerase eta (Poleta) in the replicative bypass of m6G lesions in DNA. The formation of MNNG-induced mutations is almost abolished in the rad30Delta pol32Delta double mutant of yeast, which lacks the RAD30 gene that encodes Poleta and the Pol32 subunit of DNA polymerase delta (Poldelta). Although Poldelta can function in the mutagenic bypass of m6G lesions, our biochemical studies indicate that Poleta is much more efficient in replicating through m6G than Poldelta. Both Poleta and Poldelta insert a C or a T residue opposite from m6G; Poleta, however, is more accurate, as it inserts a C about twice as frequently as Poldelta. Alkylating agents are used in the treatment of malignant tumors, including lymphomas, brain tumors, melanomas, and gastrointestinal carcinomas, and the clinical effectiveness of these agents derives at least in part from their ability to form m6G in DNA. Inactivation of Poleta could afford a useful strategy for enhancing the effectiveness of these agents in cancer chemotherapy.

Alkylating Agents↗
PubMed1999

Fidelity and processivity of Saccharomyces cerevisiae DNA polymerase eta.

The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol eta, that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA. To better understand the role of pol eta in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates. Steady-state kinetic analyses of deoxynucleotide incorporation indicate that pol eta has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10(-2) to 10(-3). Also pol eta has a low processivity, incorporating only a few nucleotides before dissociating. We suggest that pol eta's low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors.

DNA Damage↗
PubMed1999

Requirement of yeast SGS1 and SRS2 genes for replication and transcription.

The SGS1 gene of the yeast Saccharomyces cerevisiae encodes a DNA helicase with homology to the human Bloom's syndrome gene BLM and the Werner's syndrome gene WRN. The SRS2 gene of yeast also encodes a DNA helicase. Simultaneous deletion of SGS1 and SRS2 is lethal in yeast. Here, using a conditional mutation of SGS1, it is shown that DNA replication and RNA polymerase I transcription are drastically inhibited in the srs2Delta sgs1-ts strain at the restrictive temperature. Thus, SGS1 and SRS2 function in DNA replication and RNA polymerase I transcription. These functions may contribute to the various defects observed in Werner's and Bloom's syndromes.

Bloom Syndrome↗
PubMed1999

Synergistic interaction between yeast nucleotide excision repair factors NEF2 and NEF4 in the binding of ultraviolet-damaged DNA.

Saccharomyces cerevisiae RAD4, RAD7, RAD16, and RAD23 genes function in the nucleotide excision repair (NER) of ultraviolet light (UV)-damaged DNA. Previous biochemical studies have shown that the Rad4 and Rad23 proteins are associated in a stoichiometric complex named NEF2, and the Rad7 and Rad16 proteins form another stoichiometric complex named NEF4. While NEF2 is indispensable for the incision of UV-damaged DNA in the in vitro reconstituted system, NEF4 stimulates the incision reaction. Both NEF2 and NEF4 bind UV-damaged DNA, which raises the intriguing possibility that these two complexes cooperate to achieve the high degree of specificity for DNA damage demarcation required for nucleotide excision repair in vivo. Consistent with this hypothesis, we find that NEF2 and NEF4 bind in a synergistic fashion to UV-damaged DNA in a reaction that is dependent on ATP. We also purify the Rad7 protein and show that it binds DNA but has no preference for UV-damaged DNA. Rad7 physically interacts with NEF2, suggesting a role for Rad7 in linking NEF2 with NEF4.

Adenosine Triphosphatases↗
PubMed1999

hRAD30 mutations in the variant form of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is an autosomal recessive disease characterized by a high incidence of skin cancers. Yeast RAD30 encodes a DNA polymerase involved in the error-free bypass of ultraviolet (UV) damage. Here it is shown that XP variant (XP-V) cell lines harbor nonsense or frameshift mutations in hRAD30, the human counterpart of yeast RAD30. Of the eight mutations identified, seven would result in a severely truncated hRad30 protein. These results indicate that defects in hRAD30 cause XP-V, and they suggest that error-free replication of UV lesions by hRad30 plays an important role in minimizing the incidence of sunlight-induced skin cancers.

Alleles↗
PubMed1999

Requirement of DNA polymerase activity of yeast Rad30 protein for its biological function.

The RAD30 gene of Saccharomyces cerevisiae encodes a DNA polymerase, Poleta. The Rad30 protein shares homology with the yeast Rev1 and the Escherichia coli DinB and UmuC proteins. Although these proteins contain several highly conserved motifs, only Rad30 has been shown to possess a DNA polymerase activity. To determine whether the DNA polymerase activity of Rad30 was essential for its biological function, we made a mutation in the highly conserved SIDE sequence in Rad30, in which the aspartate and glutamate residues have each been changed to alanine. The mutant Rad30 protein lacks the DNA polymerase activity, and the mutant gene does not complement the rad30Delta mutation. These findings indicate that DNA polymerase activity is indispensable for the biological function of RAD30.

Amino Acid Sequence↗