Conformational trapping of mismatch recognition complex MSH2/MSH3 on repair-resistant DNA loops.

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

Discrete interactions between bacteriophage T7 primase-helicase and DNA polymerase drive the formation of a priming complex containing two copies of DNA polymerase.

Replisomes are multiprotein complexes that coordinate the synthesis of leading and lagging DNA strands to increase the replication efficiency and reduce DNA strand breaks caused by stalling of replication forks. The bacteriophage T7 replisome is an economical machine that requires only four proteins for processive, coupled synthesis of two DNA strands. Here we characterize a complex between T7 primase-helicase and DNA polymerase on DNA that was trapped during the initiation of Okazaki fragment synthesis from an RNA primer. This priming complex consists of two DNA polymerases and a primase-helicase hexamer that assemble on the DNA template in an RNA-dependent manner. The zinc binding domain of the primase-helicase is essential for trapping the RNA primer in complex with the polymerase, and a unique loop located on the thumb of the polymerase also stabilizes this primer extension complex. Whereas one of the polymerases engages the primase-helicase and RNA primer on the lagging strand of a model replication fork, the second polymerase in the complex is also functional and can bind a primed template DNA. These results indicate that the T7 primase-helicase specifically engages two copies of DNA polymerase, which would allow the coordination of leading and lagging strand synthesis at a replication fork. Assembly of the T7 replisome is driven by intimate interactions between the DNA polymerase and multiple subunits of the primase-helicase hexamer.

ATP hydrolysis by RAD50 protein switches MRE11 enzyme from endonuclease to exonuclease.

MRE11-RAD50 is a key early response protein for processing DNA ends of broken chromosomes for repair, yet how RAD50 nucleotide dynamics regulate MRE11 nuclease activity is poorly understood. We report here that ATP binding and ATP hydrolysis cause a striking butterfly-like opening and closing of the RAD50 subunits, and each structural state has a dramatic functional effect on MRE11. RAD50-MRE11 has an extended conformation in solution when MRE11 is an active nuclease. However, ATP binding to RAD50 induces a closed conformation, and in this state MRE11 is an endonuclease. ATP hydrolysis opens the RAD50-MRE11 complex, and MRE11 maintains exonuclease activity. Thus, ATP hydrolysis is a molecular switch that converts MRE11 from an endonuclease to an exonuclease. We propose a testable model in which the open-closed transitions are used by RAD50-MRE11 to discriminate among DNA ends and drive the choice of recombination pathways.

Application of 3D Scanning Method to Assess Mounting Holes' Shape Instability of Pinewood.

Swelling and shrinkage anisotropy affect the susceptibility to an assembly of wooden elements by changing designed clearances or interference fits. This work described the new method to measure mounting holes' moisture-induced shape instability and its verification using three sets of twin samples made of Scots pinewood. Each set of samples contained a pair with different grain patterns. All samples were conditioned under reference conditions (relative air humidity-RH = 60% and temperature 20 °C), and their moisture content (MC) reached equilibrium (10.7 ± 0.1%). On the side of each sample, the seven mounting holes of 12 mm in diameter were drilled. Immediately after drilling, Set 1 was used to measure the effective hole diameter with 15 cylindrical plug-gauges with diameters of 0.05 mm step, while Set 2 and Set 3 were separately re-seasoned by six months in two extreme conditions. Set 2 was conditioned with air at 85% RH (reached an equilibrium MC of 16.6 ± 0.5%), while Set 3 was exposed to air at 35% RH (reached an equilibrium MC of 7.6 ± 0.1%). Results of the plug gauge tests highlighted that holes in the samples subjected to swelling (Set 2) increased an effective diameter in the range of 12.2-12.3 mm (1.7-2.5%), while samples subjected to shrinking (Set 3) reduced the effective diameter to 11.9-11.95 mm (0.8-0.4%). To accurately reproduce the complex shape of the deformation, gypsum casts of holes were made. The 3D optical scanning method was used to read the gypsum casts' shape and dimensions. The 3D surface map of deviations analysis provided more detailed information than the plug-gauge test results. Both the shrinking and swelling of the samples changed the shapes and sizes of the holes, but shrinking reduced the effective diameter of the hole more than swelling increased it. The moisture-induced changes in the shape of holes are complex: the holes ovalized with a different range, depending on the wood grain pattern and hole depth, and were slightly extended in diameter at the bottom. Our study provides a new way to measure 3D hole initial shape changes in wooden elements during desorption and absorption.

Characteristics of Chitosan Films with the Bioactive Substances-Caffeine and Propolis.

Chitosan is a natural and biodegradable polymer with promising potential for biomedical applications. This study concerns the production of chitosan-based materials for future use in the medical industry. Bioactive substances-caffeine and ethanolic propolis extract (EEP)-were incorporated into a chitosan matrix to increase the bioactivity of the obtained films and improve their mechanical properties. Acetic and citric acids were used as solvents in the production of the chitosan-based films. The obtained materials were characterized in terms of their antibacterial and antifungal activities, as well as their mechanical properties, including tensile strength and elongation at break. Moreover, the chemical structures and surface morphologies of the films were assessed. The results showed that the solution consisting of chitosan, citric acid, caffeine, and EEP exhibited an excellent antiradical effect. The activity of this solution (99.13%) was comparable to that of the standard antioxidant Trolox (92.82%). In addition, the film obtained from this solution showed good antibacterial activity, mainly against Escherichia coli and Enterococcus faecalis. The results also revealed that the films produced with citric acid exhibited higher activity levels against pathogenic bacteria than the films obtained with acetic acid. The antimicrobial effect of the chitosan-based films could be further enhanced by adding bioactive additives such as caffeine and propolis extract. The mechanical tests showed that the solvents and additives used affected the mechanical properties of the films obtained. The film produced from chitosan and acetic acid was characterized by the highest tensile strength value (46.95 MPa) while the chitosan-based film with citric acid showed the lowest value (2.28 MPa). The addition of caffeine and propolis to the film based on chitosan with acetic acid decreased its tensile strength while in the case of the chitosan-based film with citric acid, an increase in strength was observed. The obtained results suggested that chitosan films with natural bioactive substances can be a promising alternative to the traditional materials used in the medical industry, for example, as including biodegradable wound dressings or probiotic encapsulation materials.

Chemical Composition and Related Properties of Lime (Tilia cordata Mill.) Bark and Wood as Affected by Tree Growth Conditions.

Tilia cordata Mill. is a favourite tree used in urban spaces. For this reason, it is important to know its sensitivity to environmental stress, which is particularly burdensome for vegetation in urban spaces. The aim of the study was to investigate the properties necessary to control the growth of these trees and their subsequent use, i.e., chemical properties (percentage contents of cellulose, holocellulose, lignin, pentosans and substances soluble in NaOH and EtOH) as well as the chemical elements (K, Na, Mg, Ca and Fe, Zn, Cu, Pb, Cd, B, Ni, Cr, Al, As and Hg) and selected hygroscopic properties (hysteresis and sorption isotherms). Trees of Tilia cordata Mill. growing in environments exposed to environmental stress of varying severity were examined. Regardless of the growth conditions, in terms of its chemical composition, bark differs significantly from wood, showing twice the contents of soluble substances in NaOH and lignin and half the content of polysaccharides. Growth conditions clearly affect the range of selected chemical components in bark, e.g., substances soluble in ethanol, cellulose, or lignin. The main inorganic elements in bark and wood are Na, K, Ca, Mg and Zn. In bark, a relationship was found between the content of most chemical elements and differing environmental growth conditions. It was shown that environmental stress influenced the hygroscopic properties of wood and bark, which are a consequence of the percentage of chemical components.

Effects of Harvest Maturity on the Chemical and Energetic Properties of Corn Stover Biomass Combustion.

Over the last decade, there has been increased interest in applying biomass as a raw material for producing biofuels used for thermochemical conversions. Extensive use of biomass could lead to controversial competition for arable land, water, and food; therefore, only waste materials and agricultural by-products and residues should be used to produce biofuels. One suitable by-product of agricultural production is crop residue from the harvest of maize for grain (corn stover). The harvest residues of corn stover consist of four fractions, i.e., husks, leaves, cobs, and stalks, which are structurally and morphologically distinct. The aim of the study was to determine the effect of selected maize cultivars with distinct FAO (Food and Agriculture Organization of the United Nations) earliness classifications on the chemical and energetic properties of their corn cob cores. We determined the chemical properties based on elemental analysis, and the energy properties based on the heat of combustion and calorific values. The content of ash and volatile compounds in the corn cobs were also determined. The results indicated that the heat of combustion of fresh and seasoned corn cob cores ranged from 7.62-10.79 MJ/kg and 16.19-16.53 MJ/kg, respectively. The heat of combustion and calorific value of corn cob cores in the fresh state differed significantly and were strongly correlated with maize cultivars with distinct FAO earliness.

The Effect of Chitosan Type on Biological and Physicochemical Properties of Films with Propolis Extract.

The aim of the research was to determine the influence of chitosan type and propolis extract concentration on biological and physicochemical properties of chitosan-propolis films in terms of their applicability in food packaging. The films were prepared using three types of chitosan: from crab shells, medium and high molecular weight and propolis concentration in the range of 0.75-5.0%. The prepared polysaccharide films were tested for antimicrobial properties, oxygen transmission rate (OTR) and water vapor transmission rate (WVTR). Moreover, sorption tests and structural analysis were carried out. Microbiological tests indicated the best antimicrobial activity for the film consisting of high molecular weight chitosan and 5.0% propolis extract. Both the type of chitosan and propolis concentration affected transmission parameters-OTR and WVTR. The best barrier properties were recorded for the film composed of high molecular weight chitosan and 5.0% propolis extract. The results of sorption experiments showed a slight influence of chitosan type and a significant effect of propolis extract concentration on equilibrium moisture content of tested films. Moreover, propolis extract concentration affected monolayer water capacity (Mm) estimated using the Guggenheim, Anderson and de Boer (GAB) sorption model. The obtained results indicate that chitosan films with an addition of propolis extract are promising materials for food packaging applications, including food containing probiotic microorganisms.

Moisture-Dependent Strength Properties of Thermally-Modified Fraxinus excelsior Wood in Compression.

European ash (Fraxinus excelsior L.) is one of the species commonly used for wood thermal modification that improves its performance. The presented research aimed to investigate a moisture-dependent strength anisotropy of thermally-modified European ash in compression. Wood samples were modified at 180 °C and 200 °C. Their mechanical parameters were determined in the principal anatomical directions under dry (moisture content of 3%) and wet (moisture content above fibre saturation point) conditions. Effect of heat treatment temperature and moisture content on the ash wood mechanical parameters concerning each anatomical direction were determined. The results show that thermal treatment kept the intrinsic anisotropy of wood mechanical properties. It decreased wood hygroscopicity, which resulted in improved strength and elasticity measured for wet wood when compared to untreated and treated samples. Higher treatment temperature (200 °C) increased wood elasticity in compression in all the anatomical directions despite wood moisture content during the measurements. Multivariate analysis revealed that the modification temperature significantly affected the modulus of elasticity perpendicular to the grain, while in the case of compression strength, the statistically significant effect was observed only parallel to the grain. The results obtained can be useful from an industrial perspective and can serve as part of a database for further modelling purposes.

Function of Rad17/Mec3/Ddc1 and its partial complexes in the DNA damage checkpoint.

The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans) is an early response factor to DNA damage in a signal transduction pathway leading to the activation of the checkpoint system and eventually to cell cycle arrest. These subunits show structural similarities with the replication clamp PCNA and indeed, it was demonstrated in vitro that Rad17/3/1 could be loaded onto DNA by checkpoint specific clamp loader Rad24-RFC, analogous to the PCNA-RFC clamp-clamp loader system. We have studied the interactions between the checkpoint clamp subunits and the activity of partial clamp complexes. We find that none of the possible partial complexes makes up a clamp that can be loaded onto DNA by Rad24-RFC. In agreement, overexpression of DDC1 or RAD17 in a MEC3Delta strain, or of MEC3 or RAD17 in a DDC1Delta strain shows no rescue of damage sensitivity.

Overproduction and purification of RFC-related clamp loaders and PCNA-related clamps from Saccharomyces cerevisiae.

The replication clamp PCNA and its loader RFC (Replication Factor C) are central factors required for processive replication and coordinated DNA repair. Recently, several additional related clamp loaders have been identified. These alternative clamp loaders contain the small Rfc2-5 subunits of RFC, but replace the large Rfc1 subunit by a pathway-specific alternative large subunit, Rad24 for the DNA damage checkpoint, Ctf18 for the establishment of sister chromatid cohesion, and Elg1 for a general function in chromosome stability. In order to define biochemical functions for these loaders, the loaders were overproduced in yeast and purified at a milligram scale. To aid in purification, the large subunit of each clamp loader was fused to a GST-tag that, after purification could be easily removed by a rhinoviral protease. This methodology yielded all clamp loaders in high yield and with high enzymatic activity. The yeast 9-1-1 checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, was overproduced and purified in a similar manner.

Architecture of bacterial replication initiation complexes: orisomes from four unrelated bacteria.

Bacterial chromosome replication is mediated by single initiator protein, DnaA, that interacts specifically with multiple DnaA boxes located within the origin (oriC). We compared the architecture of the DnaA-origin complexes of evolutionarily distantly related eubacteria: two Gram-negative organisms, Escherichia coli and Helicobacter pylori, and two Gram-positive organisms, Mycobacterium tuberculosis and Streptomyces coelicolor. Their origins vary in size (from approx. 200 to 1000 bp) and number of DnaA boxes (from 5 to 19). The results indicate that: (i) different DnaA proteins exhibit various affinities toward single DnaA boxes, (ii) spatial arrangement of two DnaA boxes is crucial for the H. pylori and S. coelicolor DnaA proteins, but not for E. coli and M. tuberculosis proteins, and (iii) the oriC regions are optimally adjusted to their cognate DnaA proteins. The primary functions of multiple DnaA boxes are to determine the positioning and order of assembly of the DnaA molecules. Gradual transition from the sequence-specific binding of the DnaA protein to binding through co-operative protein-protein interactions seems to be a common conserved strategy to generate oligomeric initiator complexes bound to multiple sites within the chromosomal, plasmid and virial origins.

The PCNA-RFC families of DNA clamps and clamp loaders.

The proliferating cell nuclear antigen PCNA functions at multiple levels in directing DNA metabolic pathways. Unbound to DNA, PCNA promotes localization of replication factors with a consensus PCNA-binding domain to replication factories. When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling. Its modification by ubiquitin directs the cellular response to DNA damage. The ring-like PCNA homotrimer encircles double-stranded DNA and slides spontaneously across it. Loading of PCNA onto DNA at template-primer junctions is performed in an ATP-dependent process by replication factor C (RFC), a heteropentameric AAA+ protein complex consisting of the Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits. Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E. coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44/62). Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA. This stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes. Indeed, alternative RFC complexes consisting of the small RFC subunits together with an alternative Rfc1-like subunit have been identified. A complex required for the DNA damage checkpoint contains the Rad24 subunit, a complex required for sister chromatid cohesion contains the Ctf18 subunit, and a complex that aids in genome stability contains the Elg1 subunit. Only the RFC-Rad24 complex has a known associated clamp, a heterotrimeric complex consisting of Rad17, Mec3, and Ddc1. The other putative clamp loaders could either act on clamps yet to be identified or act on the two known clamps.

[Responsibilities of enterprises introducing new dangerous chemical substances and preparations]. / Obowiazki podmiotów gospodarczych wprowadzajacych do obrotu substancje lub preparaty niebezpieczne.

The paper reviews the responsibilities of producers, importers and distributors set in a new Act of January 2001 on chemical substances and preparations (Off. J. 2001, No. 11, item 84, with subsequent amendments). This Act together with executive provisions is aimed at harmonizing Polish legislation with EU requirements. The Act sets conditions, restriction and bans of production placing on the market and use of chemical substances and preparations in order to protect human health and environment against their harmful effects. The Act together with a number of executive provisions render those who introduce dangerous chemicals and chemical preparations, including distributors responsible for: classification and labelling of dangerous chemical substances and preparations; possessing, making available and up-dating safety data sheets; supplying packages containing certain dangerous substances with child-proof fastenings; notifying the Inspector for Chemical Substances and Preparations about placing a dangerous preparation on the market; notifying the Inspector about a new substance and conducting required studies; being properly qualified to handle dangerous substances. The Act strictly defines the term "placing a substance or a preparation on the market"--it means making a substance or a preparation available to third parties on the territory of The Republic of Poland, territories of the Member States of the European Union or the territory of Iceland, Liechtenstein and Norway, unless the Act provides otherwise; it also means introduction of a substance or a preparation from outside of the territory referred to above on the customs territory of The Republic of Poland, or that of the member states of the European Union and other states listed above. In addition, some of the responsibilities defined by the provisions of the law on chemical substances and preparations are also applicable to handling of biocidals, which are classified as dangerous substances. The Act also defines the terms "substance" and "chemical preparation" and sets the rules for classification and labelling of dangerous chemical substances and preparations (criteria for classification, rules for labelling, introduces the official classification and labelling of certain substances in the "list of dangerous substances"). The Act identifies methods to be used in the tests of physico-chemical properties, toxicity and ecotoxicity of chemical substances and preparations to meet the legal requirements and sets criteria to be followed by institutions involved in such testing.

Analysis of protein-DNA interactions using surface plasmon resonance.

Protein-DNA interactions are required for access and protection of the genetic information within the cell. Historically these interactions have been studied using genetic, biochemical, and structural methods resulting in qualitative or semiquantitative interaction data. In the future the focus will be on high quality quantitative data to model a huge number of interactions forming a specific network in system biology approaches. Toward this aim, BIAcore introduced in 1990 the first commercial machine that uses surface plasmon resonance (SPR) to study the real-time kinetics of biomolecular interactions. Since then systems have been developed to allow for robust analysis of a multitude of protein-DNA interactions. Here we provide a detailed guide for protein-DNA interaction analysis using the BIAcore, starting with a description of the SPR technology, giving recommendations on preliminary studies, and finishing with extensive information on quantitative and qualitative data analysis. One focus is on cooperative protein-DNA interactions, where proteins interact with each other to modulate their binding specificity or affinity. The BIAcore has been used for the last 14 years to study protein-DNA interactions; our literature review focuses on some high quality studies describing a wide range of experimental uses, covering simple 1 : 1 interactions, analysis of complicated multiprotein-DNA interaction systems, and analytical uses.

Clamping the Mec1/ATR checkpoint kinase into action.

The yeast checkpoint protein kinase Mec1, the ortholog of human ATR, is the essential upstream regulator of the cell cycle checkpoint in response to DNA damage and to stalling of DNA replication forks. The activity of Mec1/ATR is not directly regulated by the DNA substrates that signal checkpoint activation. Rather the signal appears to be transduced to Mec1 by factors that interact with the signaling DNA substrates. One of these factors, the DNA damage checkpoint clamp Rad17-Mec3-Ddc1 (human 9-1-1) is loaded onto gapped DNA resulting from the partial repair of DNA damage, and the Ddc1 subunit of this complex activates Mec1. In vertebrate cells, the TopBP1 protein (Cut5 in S. pombe and Dpb11 in S. cervisiae) that is also required for establishment of the replication fork, functions during replication fork dysfunction to activate ATR. Both mechanisms of activation generally upregulate the kinase activity towards all downstream targets.

The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint.

Yeast Mec1/Ddc2 protein kinase, the ortholog of human ATR/ATRIP, plays a central role in the DNA damage checkpoint. The PCNA-like clamp Rad17/Mec3/Ddc1 (the 9-1-1 complex in human) and its loader Rad24-RFC are also essential components of this signal transduction pathway. Here we have studied the role of the clamp in regulating Mec1, and we delineate how the signal generated by DNA lesions is transduced to the Rad53 effector kinase. The checkpoint clamp greatly activates the kinase activity of Mec1, but only if the clamp is appropriately loaded upon partial duplex DNA. Activated Mec1 phosphorylates the Ddc1 and Mec3 subunits of the clamp, the Rad24 subunit of the loader, and the Rpa1 and Rpa2 subunits of RPA. Phosphorylation of Rad53, and of human PHAS-1, a nonspecific target, also requires a properly loaded clamp. Phosphorylation and binding studies with individual clamp subunits indicate that the Ddc1 subunit mediates the functional interactions with Mec1.

Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions.

The heterotrimeric checkpoint clamp comprises the Saccharomyces cerevisiae Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans). This DNA damage response factor is loaded onto DNA by the Rad24-RFC (replication factor C-like complex with Rad24) clamp loader and ATP. Although Rad24-RFC alone does not bind to naked partial double-stranded DNA, coating of the single strand with single-stranded DNA-binding protein RPA (replication protein A) causes binding of Rad24-RFC via interactions with RPA. However, RPA-mediated binding is abrogated when the DNA is coated with RPA containing a rpa1-K45E (rfa1-t11) mutation. These properties allowed us to determine the role of RPA in clamp-loading specificity. The Rad17/3/1 clamp is loaded with comparable efficiency onto naked primer/template DNA with either a 3'-junction or a 5'-junction. Remarkably, when the DNA was coated with RPA, loading of Rad17/3/1 at 3'-junctions was completely inhibited, thereby providing specificity to loading at 5'-junctions. However, Rad17/3/1 loaded at 5'-junctions can slide across double-stranded DNA to nearby 3'-junctions and thereby affect the activity of proteins that act at 3'-termini. These studies show a unique specificity of the checkpoint loader for 5'-junctions of RPA-coated DNA. The implications of this specificity for checkpoint function are discussed.

Cluster of DnaA boxes involved in regulation of Streptomyces chromosome replication: from in silico to in vivo studies.

In Streptomyces coelicolor, replication is initiated by the DnaA protein in the centrally located oriC region and proceeds bidirectionally until the replication forks reach the ends of the linear chromosome. We identified three clusters of DnaA boxes (H69, H24, and D78) which are in a relatively short segment of the chromosome centered on the oriC region. Of the clusters analyzed, D78 exhibited the highest affinity for the DnaA protein; the affinity of DnaA for the D78 cluster was about eightfold higher than the affinity for oriC. The high-affinity DnaA boxes appear to be involved in the control of chromosome replication. Deletion of D78 resulted in more frequent chromosome replication (an elevated ratio of origins to chromosome ends was observed) and activated aerial mycelium formation, leading to earlier colony maturation. In contrast, extra copies of D78 (delivered on a plasmid) caused slow colony growth, presumably because of a reduction in the frequency of initiation of chromosome replication. This suggests that the number of high-affinity DnaA boxes is relatively constant in hyphal compartments and that deletion of D78 therefore permits an increased copy number of either the chromosomal origin region or a plasmid harboring the D78 cluster. This system conceivably influences the timing of decisions to initiate aerial mycelial formation and sporulation.

Proliferating cell nuclear antigen promotes translesion synthesis by DNA polymerase zeta.

DNA polymerase zeta (Pol zeta), a heterodimer of Rev3 and Rev7, is essential for DNA damage provoked mutagenesis in eukaryotes. DNA polymerases that function in a processive complex with the replication clamp proliferating cell nuclear antigen (PCNA) have been shown to possess a close match to the consensus PCNA-binding motif QxxLxxFF. This consensus motif is lacking in either subunit of Pol zeta, yet its activity is stimulated by PCNA. In particular, translesion synthesis of UV damage-containing DNA is dramatically stimulated by PCNA such that translesion synthesis rates are comparable with replication rates by Pol zeta on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol zeta. Efficient PCNA stimulation required that PCNA was prevented from sliding off the damage-containing model oligonucleotide template-primer through the use of biotin-streptavidin bumpers or other blocks. Under those experimental conditions, facile bypass of the abasic site was also detected by DNA polymerase delta or eta (Rad30). The yeast DNA damage checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, and an ortholog of human 9-1-1, has been implicated in damage-induced mutagenesis. However, this checkpoint clamp did not stimulate translesion synthesis by Pol zeta or by DNA polymerase delta.