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Biomedical subjects

L Prakash

Publications and source records attributed to L Prakash.

At least 19 recordsLinked to original sources
PubMed2006

Changing trends in US injury profiles: revisiting non-fatal occupational injury statistics.

The purpose of this paper is to review the current trends in non-fatal injury profiles of workers in the United States. It is generally accepted that occupational injury and illness rates are affected by many factors, such as the amount and quality of training, employee turnover rates, work experience, extent of mechanization and automation, job-related parameters, and worker gender. In the last decade, not only have the technologies used in the workplace changed significantly, there has been a greater awareness among employers and employees as to the importance of containing work injuries. Additionally, the extent of outsourcing for labor-intensive jobs has increased dramatically owing to cheaper labor costs in places such as China and Mexico. These changes have affected the manufacturing sector of US industry more than any other sector. How these changes have influenced the injury and illness profiles of the American worker is of considerable interest given the increased attention paid to work-workplace design, injury hazard control, and ergonomics in general. In this paper, we compare the injury and illness profiles of US workers separated by nearly a decade. The trends from early 1990s are compared to those from early 2000s. Data from the Bureau of Labor Statistics were used to compile the injury statistics. The results of our comparison show that while the absolute numbers of work-related injuries and illnesses have declined over the last 10 years, the basic trends associated with different factors remain almost unchanged. The reasons for this decline are discussed in this paper.

Back Injuries↗
PubMed2001

Yeast DNA polymerase eta utilizes an induced-fit mechanism of nucleotide incorporation.

DNA polymerase eta (Poleta) is unique among eukaryotic DNA polymerases in its proficient ability to replicate through distorting DNA lesions, and Poleta synthesizes DNA with a low fidelity. Here, we use pre-steady-state kinetics to investigate the mechanism of nucleotide incorporation by Poleta and show that it utilizes an induced-fit mechanism to selectively incorporate the correct nucleotide. Poleta discriminates poorly between the correct and incorrect nucleotide at both the initial nucleotide binding step and at the subsequent induced-fit conformational change step, which precedes the chemical step of phosphodiester bond formation. This property enables Poleta to bypass lesions with distorted DNA geometries, and it bestows upon the enzyme a low fidelity.

DNA↗
PubMed2001

Targeting of human DNA polymerase iota to the replication machinery via interaction with PCNA.

Human DNA polymerase iota (hPoliota) promotes translesion synthesis by inserting nucleotides opposite highly distorting or noninstructional DNA lesions. Here, we provide evidence for the physical interaction of hPoliota with proliferating cell nuclear antigen (PCNA), and show that PCNA, together with replication factor C (RFC) and replication protein A (RPA), stimulates the DNA synthetic activity of hPoliota. In the presence of these protein factors, on undamaged DNA, the efficiency (V(max)/K(m)) of correct nucleotide incorporation by hPoliota is increased approximately 80-150-fold, and this increase in efficiency results from a reduction in the apparent K(m) for the nucleotide. PCNA, RFC, and RPA also stimulate nucleotide incorporation opposite the 3'-T of the (6) thymine-thymine (T-T) photoproduct and opposite an abasic site. The interaction of hPoliota with PCNA implies that the targeting of this polymerase to the replication machinery stalled at a lesion site is achieved via this association.

Base Sequence↗
PubMed2001

Fidelity and damage bypass ability of Schizosaccharomyces pombe Eso1 protein, comprised of DNA polymerase eta and sister chromatid cohesion protein Ctf7.

DNA polymerase eta (Poleta) functions in error-free bypass of ultraviolet light-induced DNA lesions, and mutational inactivation of Poleta in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum (XPV). Both Saccharomyces cerevisiae and human Poleta efficiently insert two adenines opposite the two thymines of a cyclobutane pyrimidine dimer. Interestingly, in the fission yeast Schizosaccharomyces pombe, the eso1(+) encoded protein is comprised of two domains, wherein the NH(2) terminus is highly homologous to Poleta, and the COOH terminus is highly homologous to the S. cerevisiae Ctf7 protein which is essential for the establishment of sister chromatid cohesion during S phase. Here we characterize the DNA polymerase activity of S. pombe GST-Eso1 fusion protein and a truncated version containing only the Poleta domain. Both proteins exhibit a similar DNA polymerase activity with a low processivity, and steady-state kinetic analyses show that on undamaged DNA, both proteins misincorporate nucleotides with frequencies of approximately 10(-2) to 10(-3). We also examine the two proteins for their ability to replicate a cyclobutane pyrimidine dimer-containing DNA template and find that both proteins replicate through the lesion equally well. Thus, fusion with Ctf7 has no significant effect on the DNA replication or damage bypass properties of Poleta. The possible role of Ctf7 fusion with Poleta in the replication of Cohesin-bound DNA sequences is discussed.

Acetyltransferases↗
PubMed2001

Accuracy of lesion bypass by yeast and human DNA polymerase eta.

DNA polymerase eta (Pol eta) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Pol eta in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Pol eta replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Pol eta, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of approximately 10(-2) to 10(-3) opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Pol eta in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Pol eta to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Pol eta extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of approximately 10(-2) to 10(-3) relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Pol eta would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3'-->5' exonuclease. Replication through a TT dimer by Pol eta then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

DNA Damage↗
PubMed2001

Roles of yeast DNA polymerases delta and zeta and of Rev1 in the bypass of abasic sites.

Abasic (AP) sites are one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA machinery. Here we show efficient bypass of an AP site by the combined action of yeast DNA polymerases delta and zeta. In this reaction, Poldelta inserts an A nucleotide opposite the AP site, and Polzeta subsequently extends from the inserted nucleotide. Consistent with these observations, sequence analyses of mutations in the yeast CAN1s gene indicate that A is the nucleotide inserted most often opposite AP sites. The nucleotides C, G, and T are also incorporated, but much less frequently. Enzymes such as Rev1 and Poleta may contribute to the insertion of these other nucleotides; the predominant role of Rev1 in AP bypass, however, is likely to be structural. Steady-state kinetic analyses show that Polzeta is highly inefficient in incorporating nucleotides opposite the AP site, but it efficiently extends from nucleotides, particularly an A, inserted opposite this lesion. Thus, in eukaryotes, bypass of an AP site requires the sequential action of two DNA polymerases, wherein the extension step depends solely upon Polzeta, but the insertion step can be quite varied, involving not only the predominant action of the replicative DNA polymerase, Poldelta, but also the less prominent role of various translesion synthesis polymerases.

Base Sequence↗
PubMed2001

Structure of the catalytic core of S. cerevisiae DNA polymerase eta: implications for translesion DNA synthesis.

DNA polymerase eta is unique among eukaryotic polymerases in its proficient ability to replicate through a variety of distorting DNA lesions. We report here the crystal structure of the catalytic core of S. cerevisiae DNA polymerase eta, determined at 2.25A resolution. The structure reveals a novel polydactyl right hand-shaped molecule with a unique polymerase-associated domain. We identify the catalytic residues and show that the fingers and thumb domains are unusually small and stubby. In particular, the unexpected absence of helices "O" and "O1" in the fingers domain suggests that openness of the active site is the critical feature which enables DNA polymerase eta to replicate through DNA lesions such as a UV-induced cis-syn thymine-thymine dimer.

Amino Acid Motifs↗
PubMed2001

Interaction with PCNA is essential for yeast DNA polymerase eta function.

In both yeast and humans, DNA polymerase (Pol) eta functions in error-free replication of ultraviolet-damaged DNA, and Poleta promotes replication through many other DNA lesions as well. Here, we present evidence for the physical and functional interaction of yeast Poleta with proliferating cell nuclear antigen (PCNA) and show that the interaction with PCNA is essential for the in vivo function of Poleta. Poleta is highly inefficient at inserting a nucleotide opposite an abasic site, but interaction with PCNA greatly stimulates its ability for nucleotide incorporation opposite this lesion. Thus, in addition to having a pivotal role in the targeting of Poleta to the replication machinery stalled at DNA lesions, interaction with PCNA would promote the bypass of certain DNA lesions.

Amino Acid Motifs↗
PubMed2001

Requirement of DNA polymerase eta for error-free bypass of UV-induced CC and TC photoproducts.

The yeast RAD30-encoded DNA polymerase eta (Poleta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Human DNA polymerase eta functions similarly in the bypass of this lesion, and mutations in human Poleta result in the cancer prone syndrome, the variant form of xeroderma pigmentosum. UV light, however, also elicits the formation of cis-syn cyclobutane dimers and (6-4) photoproducts at 5'-CC-3' and 5'-TC-3' sites, and in both yeast and human DNA, UV-induced mutations occur primarily by 3' C to T transitions. Genetic studies presented here reveal a role for yeast Poleta in the error-free bypass of cyclobutane dimers and (6-4) photoproducts formed at CC and TC sites. Thus, by preventing UV mutagenesis at a wide spectrum of dipyrimidine sites, Poleta plays a pivotal role in minimizing the incidence of sunlight-induced skin cancers in humans.

Alleles↗
PubMed2001

Role of DNA polymerase eta in the bypass of a (6-4) TT photoproduct.

UV light-induced DNA lesions block the normal replication machinery. Eukaryotic cells possess DNA polymerase eta (Poleta), which has the ability to replicate past a cis-syn thymine-thymine (TT) dimer efficiently and accurately, and mutations in human Poleta result in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Here, we test Poleta for its ability to bypass a (6-4) TT lesion which distorts the DNA helix to a much greater extent than a cis-syn TT dimer. Opposite the 3' T of a (6-4) TT photoproduct, both yeast and human Poleta preferentially insert a G residue, but they are unable to extend from the inserted nucleotide. DNA Polzeta, essential for UV induced mutagenesis, efficiently extends from the G residue inserted opposite the 3' T of the (6-4) TT lesion by Poleta, and Polzeta inserts the correct nucleotide A opposite the 5' T of the lesion. Thus, the efficient bypass of the (6-4) TT photoproduct is achieved by the combined action of Poleta and Polzeta, wherein Poleta inserts a nucleotide opposite the 3' T of the lesion and Polzeta extends from it. These biochemical observations are in concert with genetic studies in yeast indicating that mutations occur predominantly at the 3' T of the (6-4) TT photoproduct and that these mutations frequently exhibit a 3' T-->C change that would result from the insertion of a G opposite the 3' T of the (6-4) TT lesion.

Animals↗
PubMed2001

Physical and functional interactions of human DNA polymerase eta with PCNA.

Human DNA polymerase eta (hPoleta) functions in the error-free replication of UV-damaged DNA, and mutations in hPoleta cause cancer-prone syndrome, the variant form of xeroderma pigmentosum. However, in spite of its key role in promoting replication through a variety of distorting DNA lesions, the manner by which hPoleta is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPoleta with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPoleta inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPoleta, and steady-state kinetic studies indicate that this stimulation accrues from an increase in the efficiency of nucleotide insertion resulting from a reduction in the apparent K(m) for the incoming nucleotide.

Amino Acid Sequence↗
PubMed2001

Requirement for yeast RAD26, a homolog of the human CSB gene, in elongation by RNA polymerase II.

Mutations in the human CSB gene cause Cockayne syndrome (CS). In addition to increased photosensitivity, CS patients suffer from severe developmental abnormalities, including growth retardation and mental retardation. Whereas a deficiency in the preferential repair of UV lesions from the transcribed strand accounts for the increased photosensitivity of CS patients, the reason for developmental defects in these individuals has remained unclear. Here we provide in vivo evidence for a role of RAD26, the counterpart of the CSB gene in Saccharomyces cerevisiae, in transcription elongation by RNA polymerase II, and in addition we show that under conditions requiring rapid synthesis of new mRNAs, growth is considerably reduced in cells lacking RAD26. These findings implicate a role for CSB in transcription elongation, and they strongly suggest that impaired transcription elongation is the underlying cause of the developmental problems in CS patients.

Cell Cycle Proteins↗
PubMed2001

3'-phosphodiesterase and 3'-->5' exonuclease activities of yeast Apn2 protein and requirement of these activities for repair of oxidative DNA damage.

In Saccharomyces cerevisiae, the AP endonucleases encoded by the APN1 and APN2 genes provide alternate pathways for the removal of abasic sites. Oxidative DNA-damaging agents, such as H(2)O(2), produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini. Such 3' termini are inhibitory to synthesis by DNA polymerases. Here, we show that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'-->5' exonuclease activities, and mutation of the active-site residue Glu59 to Ala in Apn2 inactivates both these activities. Consistent with these biochemical observations, genetic studies indicate the involvement of APN2 in the repair of H(2)O(2)-induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2. From these results, we conclude that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species.

Alanine↗
PubMed2001

Acidic residues critical for the activity and biological function of yeast DNA polymerase eta.

Rad30 is a member of the newly discovered UmuC/DinB/Rad30 family of DNA polymerases. The N-terminal regions of these proteins are highly homologous, and they contain five conserved motifs, I to V, while their C-terminal regions are quite divergent. We examined the contributions of the C-terminal and N-terminal regions of Rad30 to its activity and biological function. Although deletion of the last 54 amino acids has no effect on DNA polymerase or thymine-thymine (T-T) dimer bypass activity, this C-terminal deletion-containing protein is unable to perform its biological function in vivo. The presence of a bipartite nuclear targeting sequence within this region suggests that at least one function of this portion of Rad30 is nuclear targeting. To identify the active-site residues of Rad30 important for catalysis, we generated mutations of nine acidic residues that are invariant or highly conserved among Rad30 proteins from different eukaryotic species. Mutations of the Asp30 and Glu39 residues present in motif I and of the Asp155 residue present in motif III to alanine completely inactivated the DNA polymerase and T-T dimer bypass activities, and these mutations did not complement the UV sensitivity of the rad30Delta mutation. Mutation of Glu156 in motif III to alanine confers a large reduction in the efficiency of nucleotide incorporation, whereas the remaining five Rad30 mutant proteins retain wild-type levels of DNA polymerase and T-T dimer bypass activities. From these observations, we suggest a role for the Asp30, Glu39, and Asp155 residues in the binding of two metal ions required for the reaction of the incoming deoxynucleoside 5'-triphosphate with the 3'-hydroxyl in the primer terminus, while Glu156 may participate in nucleotide binding.

Alanine↗
PubMed2000

Inefficient bypass of an abasic site by DNA polymerase eta.

DNA polymerase eta (Pol eta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately, and inactivation of Pol eta in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Also, Pol eta bypasses the 8-oxoguanine lesion efficiently by predominantly inserting a C opposite this lesion, and it bypasses the O(6)-methylguanine lesion by inserting a C or a T. To further assess the range of DNA lesions tolerated by Pol eta, here we examine the bypass of an abasic site, a prototypical noninstructional lesion. Steady-state kinetic analyses show that both yeast and human Pol eta are very inefficient in both inserting a nucleotide opposite an abasic site and in extending from the nucleotide inserted. Hence, Pol eta bypasses this lesion extremely poorly. These results suggest that Pol eta requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation.

DNA↗
PubMed2000

Translesion DNA synthesis by yeast DNA polymerase eta on templates containing N2-guanine adducts of 1,3-butadiene metabolites.

Yeast DNA polymerase eta can replicate through cis-syn cyclobutane pyrimidine dimers and 8-oxoguanine lesions with the same efficiency and accuracy as replication of an undamaged template. Previously, it has been shown that Escherichia coli DNA polymerases I, II, and III are incapable of bypassing DNA substrates containing N(2)-guanine adducts of stereoisomeric 1,3-butadiene metabolites. Here we showed that yeast polymerase eta replicates DNA containing the monoadducts (S)-butadiene monoepoxide and (S,S)-butadiene diolepoxide N(2)-guanines albeit at an approximately 200-300-fold lower efficiency relative to the control guanine. Interestingly, nucleotide incorporation opposite the (R)-butadiene monoepoxide and the (R,R)-butadiene diolepoxide N(2)-guanines was approximately 10-fold less efficient than incorporation opposite their S stereoisomers. Polymerase eta preferentially incorporates the correct nucleotide opposite and downstream of all four adducts, except that it shows high misincorporation frequencies for elongation of C paired with (R)-butadiene monoepoxide N(2)-guanine. Additionally, polymerase eta does not bypass the (R,R)- and (S,S)-butadiene diolepoxide N(2)-guanine-N(2)-guanine intra- strand cross-links, and replication is completely blocked just prior to the lesion. Collectively, these data suggest that polymerase eta can tolerate the geometric distortions in DNA conferred by the N(2)-guanine butadiene monoadducts but not the intrastrand cross-links.

Butadienes↗