Advanced Raman Spectroscopy Detection of Oxidative Damage in Nucleic Acid Bases: Probing Chemical Changes and Intermolecular Interactions in Guanosine at Ultralow Concentration.

DNA/RNA synthesis precursors are especially vulnerable to damage induced by reactive oxygen species occurring following oxidative stress. Guanosine triphosphates are the prevalent oxidized nucleotides, which can be misincorporated during replication, leading to mutations and cell death. Here, we present a novel method based on micro-Raman spectroscopy, combined with ab initio calculations, for the identification, detection, and quantification of oxidized nucleotides at low concentration. We also show that the Raman signature in the terahertz spectral range (<100 cm-1) contains information on the intermolecular assembly of guanine in tetrads, which allows us to further boost the oxidative damage detection limit. Eventually, we provide evidence that similar analyses can be carried out on samples in very small volumes at very low concentrations by exploiting the high sensitivity of surface-enhanced Raman scattering combined with properly designed superhydrophobic substrates. These results pave the way for employing such advanced spectroscopic methods for quantitatively sensing the oxidative damage of nucleotides in the cell.

Phosphorylation by CK2 regulates MUS81/EME1 in mitosis and after replication stress.

The MUS81 complex is crucial for preserving genome stability through the resolution of branched DNA intermediates in mitosis. However, untimely activation of the MUS81 complex in S-phase is dangerous. Little is known about the regulation of the human MUS81 complex and how deregulated activation affects chromosome integrity. Here, we show that the CK2 kinase phosphorylates MUS81 at Serine 87 in late-G2/mitosis, and upon mild replication stress. Phosphorylated MUS81 interacts with SLX4, and this association promotes the function of the MUS81 complex. In line with a role in mitosis, phosphorylation at Serine 87 is suppressed in S-phase and is mainly detected in the MUS81 molecules associated with EME1. Loss of CK2-dependent MUS81 phosphorylation contributes modestly to chromosome integrity, however, expression of the phosphomimic form induces DSBs accumulation in S-phase, because of unscheduled targeting of HJ-like DNA intermediates, and generates a wide chromosome instability phenotype. Collectively, our findings describe a novel regulatory mechanism controlling the MUS81 complex function in human cells. Furthermore, they indicate that, genome stability depends mainly on the ability of cells to counteract targeting of branched intermediates by the MUS81/EME1 complex in S-phase, rather than on a correct MUS81 function in mitosis.

A study of the impact of DNA helical rise on protein-DNA interaction.

Nucleosomes are not uniformly distributed along DNA and their positioning (termed "nucleosomal landscape") can be derived using data available for several genomes. In this study we analyzed DNA helical rise profiles through a tetranucleotide code, and we defined the nucleosomal landscape of several sequences forming dinucleosomes and of the sequences of huntingtin, myotonic dystrophy type 1 and fragile mental retardation 2 genes, which contained several repeated sequences. We also analyzed the profiles of some sequences interacting with transcription factors or with RNA polymerase II. In the genomes of Cenorhabditis elegans, Mus musculus and Homo sapiens we found profiles with extremely low helical rise values, characteristic of nucleosome free regions. We defined these regions as "holes" and found that their presence correlates with lamina associated domains sequences. Altogether, this study shows that DNA helical rise profile may have a role in gene expression modulation and in shaping chromosomal structure.

Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability.

DNA trinucleotide repeat (TNR) expansion underlies several neurodegenerative disorders including Huntington's disease (HD). Accumulation of oxidized DNA bases and their inefficient processing by base excision repair (BER) are among the factors suggested to contribute to TNR expansion. In this study, we have examined whether oxidation of the purine dNTPs in the dNTP pool provides a source of DNA damage that promotes TNR expansion. We demonstrate that during BER of 8-oxoguanine (8-oxodG) in TNR sequences, DNA polymerase ß (POL ß) can incorporate 8-oxodGMP with the formation of 8-oxodG:C and 8-oxodG:A mispairs. Their processing by the OGG1 and MUTYH DNA glycosylases generates closely spaced incisions on opposite DNA strands that are permissive for TNR expansion. Evidence in HD model R6/2 mice indicates that these DNA glycosylases are present in brain areas affected by neurodegeneration. Consistent with prevailing oxidative stress, the same brain areas contained increased DNA 8-oxodG levels and expression of the p53-inducible ribonucleotide reductase. Our in vitro and in vivo data support a model where an oxidized dNTPs pool together with aberrant BER processing contribute to TNR expansion in non-replicating cells.

Type and frequency of MUTYH variants in Italian patients with suspected MAP: a retrospective multicenter study.

To determine prevalence, spectrum and genotype-phenotype correlations of MUTYH variants in Italian patients with suspected MAP (MUTYH-associated polyposis), a retrospective analysis was conducted to identify patients who had undergone MUTYH genetic testing from September 2002 to February 2014. Results of genetic testing and patient clinical characteristics were collected (gender, number of polyps, age at polyp diagnosis, presence of colorectal cancer (CRC) and/or other cancers, family data). The presence of large rearrangements of the MUTYH gene was evaluated by Multiplex Ligation-dependent Probe Amplification analysis. In all, 299 patients with colorectal neoplasia were evaluated: 61.2% were males, the median age at polyps or cancer diagnosis was 50 years (16-80 years), 65.2% had <100 polyps and 51.8% had CRC. A total of 36 different MUTYH variants were identified: 13 (36.1%) were classified as pathogenetic, whereas 23 (63.9%) were variants of unknown significance (VUS). Two pathogenetic variants were observed in 78 patients (26.1%). A large homozygous deletion of exon 15 was found in one patient (<1.0%). MAP patients were younger than those with negative MUTYH testing at polyps diagnosis (P<0.0001) and at first cancer diagnosis (P=0.007). MAP patients carrying the p.Glu480del variant presented with a younger age at polyp diagnosis as compared to patients carrying p.Gly396Asp and p.Tyr179Cys variants. A high heterogeneity of MUTYH variants and a high rate of VUS were identified in a cohort of Italian patients with suspected MAP. Genotype-phenotype analysis suggests that the p.Glu480del variant is associated with a severe phenotype.

Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences.

The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase ß (POL ß) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL ß incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL ß was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences.

Genetic instability in lymphoblastoid cell lines expressing biallelic and monoallelic variants in the human MUTYH gene.

The MUTYH DNA glycosylase counteracts mutagenesis by removing adenine misincorporated opposite DNA 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated polyposis (MAP). The impact on genetic instability of the p.Tyr179Cys and p.Arg245His MUTYH variants was evaluated in lymphoblastoid cell lines (LCLs) derived from MAP patients and their relatives in comparison to wild-type LCLs. No difference in MUTYH expression was identified between wild type and LCLs with the p.Tyr179Cys, while the p.Arg245His mutation was associated with an unstable MUTYH protein. LCLs homozygous for the p.Tyr179Cys or the p.Arg245His variant contained increased DNA 8-oxodG levels and exhibited a mutator phenotype at the PIG-A gene. The extent of the increased spontaneous mutation frequency was 3-fold (range 1.6- to 4.6-fold) in four independent LCLs carrying the p.Tyr179Cys variant, while a larger increase (6-fold) was observed in two p.Arg245His LCLs. A similar hypermutability and S-phase delay following treatment with KBrO3 was observed in LCLs homozygous for either variant. When genetic instability was investigated in monoallelic p.Arg245His carriers, mutant frequencies showed an increase which is intermediate between wild-type and homozygous cells, whereas the mutator effect in heterozygous p.Tyr179Cys LCLs was similar to that in homozygotes. These findings indicate that the type of MUTYH mutation can affect the extent of genome instability associated with MUTYH inactivation. In addition, the mild spontaneous mutator phenotype observed in monoallelic carriers highlights the biological importance of this gene in the protection of the genome against endogenous DNA damage.

Understanding the role of the Q338H MUTYH variant in oxidative damage repair.

The MUTYH DNA-glycosylase is indirectly engaged in the repair of the miscoding 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxodG) lesion by removing adenine erroneously incorporated opposite the oxidized purine. Inherited biallelic mutations in the MUTYH gene are responsible for a recessive syndrome, the MUTYH-associated polyposis (MAP), which confers an increased risk of colorectal cancer. In this study, we functionally characterized the Q338H variant using recombinant proteins, as well as cell-based assays. This is a common variant among human colorectal cancer genes, which is generally considered, unrelated to the MAP phenotype but recently indicated as a low-penetrance allele. We demonstrate that the Q338H variant retains a wild-type DNA-glycosylase activity in vitro, but it shows a reduced ability to interact with the replication sensor RAD9:RAD1:HUS1 (9-1-1) complex. In comparison with Mutyh(-)(/)(-) mouse embryo fibroblasts expressing a wild-type MUTYH cDNA, the expression of Q338H variant was associated with increased levels of DNA 8-oxodG, hypersensitivity to oxidant and accumulation of the population in the S phase of the cell cycle. Thus, an inefficient interaction of MUTYH with the 9-1-1 complex leads to a repair-defective phenotype, indicating that a proper communication between MUTYH enzymatic function and the S phase checkpoint is needed for effective repair of oxidative damage.

Role of MUTYH in human cancer.

MUTYH, a human ortholog of MutY, is a post-replicative DNA glycosylase, highly conserved throughout evolution, involved in the correction of mismatches resulting from a faulty replication of the oxidized base 8-hydroxyguanine (8-oxodG). In particular removal of adenine from A:8-oxodG mispairs by MUTYH activity is followed by error-free base excision repair (BER) events, leading to the formation of C:8-oxodG base pairs. These are the substrate of another BER enzyme, the OGG1 DNA glycosylase, which then removes 8-oxodG from DNA. Thus the combined action of OGG1 and MUTYH prevents oxidative damage-induced mutations, i.e. GC->TA transversions. Germline mutations in MUTYH are associated with a recessively heritable colorectal polyposis, now referred to as MUTYH-associated polyposis (MAP). Here we will review the phenotype(s) associated with MUTYH inactivation from bacteria to mammals, the structure of the MUTYH protein, the molecular mechanisms of its enzymatic activity and the functional characterization of MUTYH variants. The relevance of these results will be discussed to define the role of specific human mutations in colorectal cancer risk together with the possible role of MUTYH inactivation in sporadic cancer.

Effect of blood storage conditions on DNA repair capacity measurements in peripheral blood mononuclear cells.

Due to the great number of genes involved in DNA repair and the interactions among the pathways responsible for the repair of different types of DNA damage, there is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacity (DRC). The use of peripheral blood mononuclear cells (PBMCs) in DRC assays is particularly useful for human monitoring studies. However, in such studies it is not always possible to collect and process samples on the same day as the blood is taken. We performed a genotype-phenotype correlation study on DRC on 225 healthy subjects. Due to the large number of blood samples to be processed, PBMCs were either isolated and cryopreserved on the same day of blood collection (day 1) or on the following day after 24h blood storage at room temperature (day 2-RT). Samples processed in different days showed a significant difference in the DRC evaluated as 8-oxoguanine glycosylase activity (OGG assay) in cell extracts (p<0.0001) and as benzo[a]pyrene diol epoxide (BPDE)-induced damage repair by the comet assay (p=0.05). No apparent effect of the blood storage conditions on the outcome of γ-ray induced H2AX phosphorylation assay was reported. These results prompted us to further analyze the effects of blood storage conditions by performing a validation study. Three blood samples were simultaneously taken from ten healthy donors, PBMCs were isolated and cryopreserved as follows: immediately after blood collection (day 1); on the following day, after blood storage at RT (day 2-RT); or after blood storage at 4°C (day 2-4°C). DRC was then evaluated using phenotypic assays. The γ-ray induced H2AX phosphorylation assay has been confirmed as the only assay that showed good reproducibility independently of the blood storage conditions. The measurement of OGG assay was most affected by the blood storage conditions.

Gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function.

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.

Reprint of: gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function.

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.

8-Oxoguanine DNA-glycosylase repair activity and expression: a comparison between cryopreserved isolated lymphocytes and EBV-derived lymphoblastoid cell lines.

Several lines of evidence suggest an association between oxidative DNA-damage repair capacity and cancer risk. In particular, a DNA-glycosylase assay for removal of 8-oxoguanine (8-oxoG) in peripheral blood mononuclear cells (PBMC) has been successfully applied to identify populations with increased risk for lung cancer and squamous cell carcinomas of head and neck. In order to verify whether EBV-transformed lymphoblastoid cell lines (LCL) are a suitable surrogate for PBMC in specific DNA-repair phenotypic assays, a validation trial was conducted. PBMC from 20 healthy subjects were collected and an aliquot was transformed with EBV to obtain LCL. The ability of cell-free extracts from both cell types to incise a 3'-fluorescently labelled duplex oligonucleotide containing a single 8-oxoG (OGG assay) was evaluated. Since this activity is mediated predominantly by OGG1, the OGG1 gene expression was also measured. 8-oxoG DNA-glycosylase activity and OGG1 expression were significantly higher (p<0.0001) in LCL than in PBMC. However, while this assay was shown to be robust and reproducible when used on PBMC (intra-assay CV=8%), a high intra-culture variability was observed with LCL (intra-culture CV=16.8%). Neither differences on OGG1 gene expression nor the cell-cycle distribution seemed to account for this variability. Inter-individual variability of OGG activity in PBMC and LCL was not associated with OGG1 gene expression. We have therefore established a non-radioactive cleavage assay that can be easily applied to measure OGG activity in human PBMC. The use of LCL for DNA-repair genotype-phenotype correlation studies seems to be inappropriate, at least with cell-free based functional assays.

MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay.

MUTYH-associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh(-/-) mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity.

Replication of 2-hydroxyadenine-containing DNA and recognition by human MutSalpha.

2-Hydroxyadenine (2-OH-A), a product of DNA oxidation, is a potential source of mutations. We investigated how representative DNA polymerases from the A, B and Y families dealt with 2-OH-A in primer extension experiments. A template 2-OH-A reduced the rate of incorporation by DNA polymerase alpha (Pol alpha) and Klenow fragment (Kf(exo-)). Two Y family DNA polymerases, human polymerase eta (Pol eta) and the archeal Dpo4 polymerase were affected differently. Bypass by Pol eta was very inefficient whereas Dpo4 efficiently replicated 2-OH-A. Replication of a template 2-OH-A by both enzymes was mutagenic and caused base substitutions. Dpo4 additionally introduced single base deletions. Thermodynamic analysis showed that 2-OH-A forms stable base pairs with T, C and G, and to a lesser extent with A. Oligonucleotides containing 2-OH-A base pairs, including the preferred 2-OH-A:T, were recognized by the human MutSalpha mismatch repair (MMR). MutSalpha also recognized 2-OH-A located in a repeat sequence that mimics a frameshift intermediate.

Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity.

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.

8-oxoguanine incorporation into DNA repeats in vitro and mismatch recognition by MutSalpha.

DNA 8-oxoguanine (8-oxoG) causes transversions and is also implicated in frameshifts. We previously identified the dNTP pool as a likely source of mutagenic DNA 8-oxoG and demonstrated that DNA mismatch repair prevented oxidation-related frameshifts in mononucleotide repeats. Here, we show that both Klenow fragment and DNA polymerase alpha can utilize 8-oxodGTP and incorporate the oxidized purine into model frameshift targets. Both polymerases incorporated 8-oxodGMP opposite C and A in repetitive DNA sequences and efficiently extended a terminal 8-oxoG. The human MutSalpha mismatch repair factor recognized DNA 8-oxoG efficiently in some contexts that resembled frameshift intermediates in the same C or A repeats. DNA 8-oxoG in other slipped/mispaired structures in the same repeats adopted configurations that prevented recognition by MutSalpha and by the OGG1 DNA glycosylase thereby rendering it invisible to DNA repair. These findings are consistent with a contribution of oxidative DNA damage to frameshifts. They also suggest how mismatch repair might reduce the burden of DNA 8-oxoG and prevent frameshift formation.

A Specific Mutational Signature Associated with DNA 8-Oxoguanine Persistence in MUTYH-defective Colorectal Cancer.

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C>T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C>T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. The occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs.

The response to oxidative stress and metallomics analysis in a twin study: The role of the environment.

Inefficient response to oxidative stress has been associated with ageing and health risk. Metals are known to inhibit DNA repair and may modify the antioxidant response. How genetic variability and lifestyle factors modulate the response to oxidative stress is poorly explored. Our study aims to disentangle the contribution of genetics and environmental exposures to oxidative stress response using data from twin pairs. The non-enzymatic antioxidant capacity (NEAC), the repair capacity of 8-oxo-7,8-dihydroguanine (OGG activity) and the levels of 12 metals were measured in blood of 64 monozygotic and 31 dizygotic twin pairs. The contributions of genetic and environmental effects were assessed using standard univariate twin modelling. NEAC and OGG activity significantly decreased with age. Gender-, age- and body mass index-associated differences were identified for some metals. Principal Component Analysis identified two groups of metals whose levels in blood were highly correlated: As, Hg, Pb, Se, Zn and Al, Co, Cr, Mn, Ni. The environmental influence was predominant on OGG activity and NEAC variance whereas for most metals the best-fitting model incorporated additive genetic and unique environmental sources of variance. NEAC and OGG activity were both inversely correlated with blood levels of various metals. The inhibition of OGG activity by Cd was largely explained by smoking. Our data show a substantial role of environmental factors in NEAC and OGG activity variance that is not explained by twins' age. Exogenous environmental factors such as metals contribute to oxidative stress by decreasing NEAC and inhibiting repair of oxidatively-induced DNA damage.

Structural and dynamic effects of single 7-hydro-8-oxoguanine bases located in a frameshift target DNA sequence.

DNA 7-hydro-8-oxoguanine (8-oxoG) is implicated in frameshift formation in an G(6) sequence of the HPRT gene in mismatch repair (MMR) defective cells. Using oligonucleotides based on this frameshift hotspot, we investigated how a single 8-oxoG modified the structural and dynamic properties of the G(6) tract. A 30 ns molecular dynamics (MD) simulation indicated compression of the minor groove in the immediate vicinity of the lesion. Fluorescence polarization anisotropy (FPA) and MD demonstrated that 8-oxoG increases DNA torsional rigidity and also constrains the movement of the single-stranded region at the single/double stranded DNA junction of model DNA replication template/primer. These constraints influenced the efficiency of primer extension by Klenow (exo(-)) DNA polymerase.