Structural insights into DNA replication and lesion bypass by Y family DNA polymerases

Y family DNA polymerases are specialized enzymes for replication through sites of DNA damage in the genome. Although the DNA damage bypass activity of these enzymes is important for genome maintenance and integrity, it is also responsible for DNA mutagenesis due to the error-prone nature of the Y family. Understanding how these enzymes select incoming nucleotides during DNA replication will give insight into their role in cancer formation, aging, and evolution. This work attempts to mechanistically explain, primarily through X-ray crystallography and enzymatic activity assays, how Y family polymerases select incoming nucleotides in various DNA replication contexts. Initially, we sought to determine how the model Y family polymerase Dpo4 differentiates between ribo and deoxyribonucleotides. Crystal structures were solved of a mutant Dpo4 enzyme (Y12A) deficient in ribonucleotide discrimination, incorporating either deoxy-adenine (dA) or ribo-adenine (rA) nucleotides opposite template thymine DNA. It was revealed that the Dpo4 Y12A mutant allowed rA incorporation by accommodating the 2’-OH group of the ribose sugar. Thus Y family polymerases block ribonucleotide incorporation during DNA replication by clashing with the 2’-OH group of ribose sugars. Next, we examined how human DNA polymerase iota (polɩ) prefers to incorporate mis-matched nucleotides opposite an undamaged thymine base. Crystal structures of polɩ in complex with template thymine DNA incorporating either correct adenine (A) or mis-matched thymine (T) or guanine (G) revealed the structural basis of error-prone replication. Correct A was destabilized by a narrow polɩ active site and mis-matched G was preferred by hydrogen bonding with glutamine59 from the finger domain. Domain swapping experiments confirmed the role of the polɩ finger domain in nucleotide selection opposite T. We then investigated how polɩ selects the correct cytosine (C) nucleotide opposite the mutagenic oxidative lesion 8-oxo-guanine. Crystal structures of polɩ in complex with 8-oxo-guanine DNA incorporating correct C or mis-matched A, T, or G revealed the structural basis of error-free replication. The narrow polɩ active site destabilizes A and G purine bases while selecting correct C due to the greatest hydrogen bonding potential with the 8-oxo-guanine Hoogsteen edge. We also show how Glu59 from the finger domain is involved in nucleotide selection and bypass activity through site-directed mutagenesis. Lastly, we examined how polɩ replicates opposite a bulky lesion produced form environmental pollution: N-[deoxyguanosin-8-yl]-1-amino-pyrene (APG). Crystal structures of polɩ in complex with APG DNA incorporating correct C or mis-matched A reveal the structural mechanism of APG replication. Correct C is preferred opposite the lesion due to Watson-Crick base pairing while mis-matched A is incorporated by base stacking above the lesion. We also demonstrate using the model Y family polymerase Dpo4, that the hydrophobic lesion interacts with protein side chains from the little finger domain, which inhibits DNA replication past the lesion site. Taken together, these results further our understanding of how Y family polymerases select incoming nucleotides and how this selection can result in error-free or error-prone replication depending on the chemical nature of the template base

Structural Basis of Error-prone Replication and Stalling at a Thymine Base by Human DNA Polymerase iota

Human DNA polymerase iota (pol iota) is a unique member of Y-family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of pol complexed with DNA containing a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson-Crick base pairing by pol. The template thymine remains in an anti conformation irrespective of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H-bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template \u27U-turn\u27 is stabilized by pol and the methyl group of the thymine template, revealing the structural basis of T stalling. Our structural and domain-swapping experiments indicate that the finger domain is responsible for pol\u27s high error rates on pyrimidines and determines the incorporation specificity

Association between physical pain and alcohol treatment outcomes: The mediating role of negative affect

OBJECTIVE: Physical pain and negative affect have been described as risk factors for alcohol use following alcohol treatment. The current study was a secondary analysis of two clinical trials for alcohol use disorder (AUD) to examine the associations between pain, negative affect and AUD treatment outcomes. METHOD: Participants included 1383 individuals from the COMBINE Study (COMBINE Study Group, 2003; 31% female, 23% ethnic minorities, average age=44.4 (SD=10.2)), a multisite combination pharmacotherapy and behavioral intervention study for AUD in the United States, and 742 individuals from the United Kingdom Alcohol Treatment Trial (UKATT Research Team, 2001; 25.9% female, 4.4% ethnic minorities, average age=41.6 (SD=10.1)) a multisite behavioral intervention study for AUD in the United Kingdom. The Form-90 was used to collect alcohol use data, the Short Form Health Survey and Quality of Life measures were used to assess pain, and negative affect was assessed using the Brief Symptom Inventory (COMBINE) and the General Health Questionnaire (UKATT). RESULTS: Pain scores were significantly associated with drinking outcomes in both datasets. Greater pain scores were associated with greater negative affect and increases in pain were associated with increases in negative affect. Negative affect significantly mediated the association between pain and drinking outcomes and this effect was moderated by social behavior network therapy (SBNT) in the UKATT study, with SBNT attenuating the association between pain and drinking. CONCLUSION: Findings suggest pain and negative affect are associated among individuals in AUD treatment and that negative affect mediated pain may be a risk factor for alcohol relapse

Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase ι

Human DNA polymerase ι (polι) is a unique member of Y-family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of polι complexed with DNA containing a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson–Crick base pairing by polι. The template thymine remains in an anti conformation irrespective of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H-bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template ‘U-turn' is stabilized by polι and the methyl group of the thymine template, revealing the structural basis of T stalling. Our structural and domain-swapping experiments indicate that the finger domain is responsible for polι's high error rates on pyrimidines and determines the incorporation specificity

Citation

Association between physical pain and alcohol treatment outcomes: The mediating role of negative affect. Journal of Consulting and Clinical Psychology, 83 (6)

Pain as a predictor of heavy drinking and any drinking lapses in the COMBINE study and the UK Alcohol Treatment Trial

Aims: To test the association between pain and heavy drinking lapses during and following treatment for alcohol use disorders (AUD). Design: Secondary data analysis of data from two clinical trials for AUD. Setting and participants: Participants included 1383 individuals from the Combined Pharmacotherapies and Behavioral Interventions (COMBINE) Study in the United States [69.0% male, 76.8% non-Hispanic White average age=44.4, standard deviation (SD)=10.2] and 742 individuals from the UK Alcohol Treatment Trial (UKATT) in the United Kingdom [74.1% male, 95.6% White, average age=41.6 (SD=10.1)]. Measurements: Form-90 (a structured assessment interview) was used to assess the primary outcome: time to first heavy drinking day. The Short Form Health Survey and Quality of Life measures were used to assess pain interference and pain intensity. Findings: Pain was a significant predictor of heavy drinking lapses during treatment in UKATT [odds ratio (OR)=1.19, 95% confidence interval (CI)=1.08, 1.32, P=0.0003] and COMBINE (OR=1.12, 95% CI=1.03, 1.21, P=0.009), and was a significant predictor of heavy drinking lapses following treatment in COMBINE (OR=1.163, 95% CI=1.15, 1.17, P&lt;0.00001). After controlling for other relapse risk factors (e.g. dependence severity, self-efficacy, temptation, psychiatric distress), pain remained a significant predictor of heavy drinking lapses during treatment in UKATT (OR=1.19, 95% CI=1.06, 1.34, P=0.004) and following treatment in COMBINE (OR=1.44, 95% CI=1.07, 1.92, P=0.01). Conclusions: Among people treated for alcohol use disorder, being in physical pain appears to predict heavy drinking lapses during or after treatment.</p