Apolipoprotein E genotype-dependent nutrigenetic effects to prebiotic inulin for modulating systemic metabolism and neuroprotection in mice via gut-brain axis.

OBJECTIVE: The goal of the study was to identify the potential nutrigenetic effects to inulin, a prebiotic fiber, in mice with different human apolipoprotein E (APOE) genetic variants. Specifically, we compared responses to inulin for the potential modulation of the systemic metabolism and neuroprotection via gut-brain axis in mice with human APOE ϵ3 and ϵ4 alleles. METHOD: We performed experiments with young mice expressing the human APOE3 (E3FAD mice and APOE4 gene (E4FAD mice). We fed mice with either inulin or control diet for 16 weeks starting from 3 months of age. We determined gut microbiome diversity and composition using16s rRNA sequencing, systemic metabolism using in vivo MRI and metabolomics, and blood-brain barrier (BBB) tight junction expression using Western blot. RESULTS: In both E3FAD and E4FAD mice, inulin altered the alpha and beta diversity of the gut microbiome, increased beneficial taxa of bacteria and elevated cecal short chain fatty acid and hippocampal scyllo-inositol. E3FAD mice had altered metabolism related to tryptophan and tyrosine, while E4FAD mice had changes in the tricarboxylic acid cycle, pentose phosphate pathway, and bile acids. Differences were found in levels of brain metabolites related to oxidative stress, and levels of Claudin-1 and Claudin-5 BBB tight junction expression. DISCUSSION: We found that inulin had many similar beneficial effects in the gut and brain for both E3FAD and E4FAD mice, which may be protective for brain functions and reduce risk for neurodegeneration. . E3FAD and E4FAD mice also had distinct responses in several metabolic pathways, suggesting an APOE-dependent nutrigenetic effects in modulating systemic metabolism and neuroprotection.

Impact of acute lymphoblastic leukemia induction therapy: findings from metabolomics on non-fasted plasma samples from a biorepository.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is among the most common cancers in children. With improvements in combination chemotherapy regimens, the overall survival has increased to over 90%. However, the current challenge is to mitigate adverse events resulting from the complex therapy. Several chemotherapies intercept cancer metabolism, but little is known about their collective role in altering host metabolism. OBJECTIVES: We profiled the metabolomic changes in plasma of ALL patients initial- and post- induction therapy. METHODS: We exploited a biorepository of non-fasted plasma samples derived from the Dana Farber Cancer Institute ALL Consortium; these samples were obtained from 50 ALL patients initial- and post-induction therapy. Plasma metabolites and complex lipids were analyzed by high resolution tandem mass spectrometry and differential mobility tandem mass spectrometry. Data were analyzed using a covariate-adjusted regression model with multiplicity adjustment. Pathway enrichment analysis and co-expression network analysis were performed to identify unique clusters of molecules. RESULTS: More than 1200 metabolites and complex lipids were identified in the total of global metabolomics and lipidomics platforms. Over 20% of those molecules were significantly altered. In the pathway enrichment analysis, lipids, particularly phosphatidylethanolamines (PEs), were identified. Network analysis indicated that the bioactive fatty acids, docosahexaenoic acid (DHA)-containing (22:6) triacylglycerols (TAGs), were decreased in the post-induction therapy. CONCLUSION: Metabolomic profiling in ALL patients revealed a large number of alterations following induction chemotherapy. In particular, lipid metabolism was substantially altered. The changes in metabolites and complex lipids following induction therapy could provide insight into the adverse events experienced by ALL patients.

APOE genotype-dependent pharmacogenetic responses to rapamycin for preventing Alzheimer's disease.

The ε4 allele of Apolipoprotein (APOE4) is the strongest genetic risk factor for Alzheimer's disease (AD), the most common form of dementia. Cognitively normal APOE4 carriers have developed amyloid beta (Aß) plaques and cerebrovascular, metabolic and structural deficits decades before showing the cognitive impairment. Interventions that can inhibit Aß retention and restore the brain functions to normal would be critical to prevent AD for the asymptomatic APOE4 carriers. A major goal of the study was to identify the potential usefulness of rapamycin (Rapa), a pharmacological intervention for extending longevity, for preventing AD in the mice that express human APOE4 gene and overexpress Aß (the E4FAD mice). Another goal of the study was to identify the potential pharmacogenetic differences in response to rapamycin between the E4FAD and E3FAD mice, the mice with human APOE ε3 allele. We used multi-modal MRI to measure in vivo cerebral blood flow (CBF), neurotransmitter levels, white matter integrity, water content, cerebrovascular reactivity (CVR) and somatosensory response; used behavioral assessments to determine cognitive function; used biochemistry assays to determine Aß retention and blood-brain barrier (BBB) functions; and used metabolomics to identify brain metabolic changes. We found that in the E4FAD mice, rapamycin normalized bodyweight, restored CBF (especially in female), BBB activity for Aß transport, neurotransmitter levels, neuronal integrity and free fatty acid level, and reduced Aß retention, which were not observe in the E3FAD-Rapa mice. In contrast, E3FAD-Rapa mice had lower CVR responses, lower anxiety and reduced glycolysis in the brain, which were not seen in the E4FAD-Rapa mice. Further, rapamycin appeared to normalize lipid-associated metabolism in the E4FAD mice, while slowed overall glucose-associated metabolism in the E3FAD mice. Finally, rapamycin enhanced overall water content, water diffusion in white matter, and spatial memory in both E3FAD and E4FAD mice, but did not impact the somatosensory responses under hindpaw stimulation. Our findings indicated that rapamycin was able to restore brain functions and reduce AD risk for young, asymptomatic E4FAD mice, and there were pharmacogenetic differences between the E3FAD and E4FAD mice. As the multi-modal MRI methods used in the study are readily to be used in humans and rapamycin is FDA-approved, our results may pave a way for future clinical testing of the pharmacogenetic responses in humans with different APOE alleles, and potentially using rapamycin to prevent AD for asymptomatic APOE4 carriers.

Association of the Social Determinants of Health With Quality of Primary Care.

PURPOSE: In primary care, there is increasing recognition of the difficulty of treating patients' immediate health concerns when their overall well-being is shaped by underlying social determinants of health. We assessed the association of social complexity factors with the quality of care patients received in primary care settings. METHODS: Eleven social complexity factors were defined using administrative data on poverty, mental health, newcomer status, and justice system involvement from the Manitoba Population Research Data Repository. We measured the distribution of these factors among primary care patients who made at least 3 visits during 2010-2013 to clinicians in Manitoba, Canada. Using generalized linear mixed modeling, we measured 26 primary care indicators to compare the quality of care received by patients with 0 to 5 or more social complexity factors. RESULTS: Among 626,264 primary care patients, 54% were living with at least 1 social complexity factor, and 4% were living with 5 or more. Social complexity factors were strongly associated with poorer outcomes with respect to primary care indicators for prevention (eg, breast cancer screening; odds ratio [OR] = 0.77; 99% CI, 0.73-0.81), chronic disease management (eg, diabetes management; OR = 0.86; 99% CI, 0.79-0.92), geriatric care (eg, benzodiazepine prescriptions; OR = 1.63; 99% CI, 1.48-1.80), and use of health services (eg, ambulatory visits; OR = 1.09; 99% CI, 1.08-1.09). CONCLUSIONS: Linking health and social data demonstrates how social determinants are associated with primary care service provision. Our findings provide insight into the social needs of primary care populations, and may support the development of focused interventions to address social complexity in primary care.

Minimal detection of nuclear mutations in XP-V and normal cells treated with oxidative stress inducing agents.

Elevated levels of reactive oxygen species (ROS) can be induced by exposure to various chemicals and radiation. One type of damage in DNA produced by ROS is modification of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG). This particular alteration to the chemistry of the base can inhibit the replication fork and has been linked to mutagenesis, cancer, and aging. In vitro studies have shown that the translesion synthesis polymerase, DNA polymerase η (pol η), is able to efficiently bypass 8-oxoG in DNA. In this study, we wanted to investigate the mutagenic effects of oxidative stress, and in particular 8-oxoG, in the presence and absence of pol η. We quantified levels of oxidative stress, 8-oxoG levels in DNA, and nuclear mutation rates. We found that most of the 8-oxoG detected were localized to the mitochondrial DNA, opposed to the nuclear DNA. We also saw a corresponding lack of mutations in a nuclear-encoded gene. This suggests that oxidative stress' primary mutagenic effects are not predominantly on genomic DNA.

Biochemical analysis of active site mutations of human polymerase η.

DNA polymerase η (pol η) plays a critical role in suppressing mutations caused by the bypass of cis-syn cyclobutane pyrimidine dimers (CPD) that escape repair. There is evidence this is also the case for the oxidative lesion 7,8-dihydro-8-oxo-guanine (8-oxoG). Both of these lesions cause moderate to severe blockage of synthesis when encountered by replicative polymerases, while pol η displays little no to pausing during translesion synthesis. However, since lesion bypass does not remove damaged DNA from the genome and can possibly be accompanied by errors in synthesis during bypass, the process is often called 'damage tolerance' to delineate it from classical DNA repair pathways. The fidelity of lesion bypass is therefore of importance when determining how pol η suppresses mutations after DNA damage. As pol η has been implicated in numerous in vivo pathways other than lesion bypass, we wanted to better understand the molecular mechanisms involved in the relatively low-fidelity synthesis displayed by pol η. To that end, we have created a set of mutant pol η proteins each containing a single amino acid substitution in the active site and closely surrounding regions. We determined overall DNA synthesis ability as well as the efficiency and fidelity of bypass of thymine-thymine CPD (T-T CPD) and 8-oxoG containing DNA templates. Our results show that several amino acids are critical for normal polymerase function, with changes in overall activity and fidelity being observed. Of the mutants that retain polymerase activity, we demonstrate that amino acids Q38, Y52, and R61 play key roles in determining polymerase fidelity, with substation of alanine causing both increases and decreases in fidelity. Remarkably, the Q38A mutant displays increased fidelity during synthesis opposite 8-oxoG but decreased fidelity during synthesis opposite a T-T CPD.

The metabolome of human milk is altered differentially by Holder pasteurization and high hydrostatic pressure processing.

The milk metabolome is composed of hundreds of molecules that can impact infant development. In preterm infants, sterilized donor milk (DM) is frequently used for their feeding. We aimed to identify differences in the metabolome of DM after two types of milk sterilization: the Holder pasteurization (HoP) and a high hydrostatic pressure (HP) processing. DM samples were sterilized by HoP (62.5°C for 30 min) or processed by HP (350 MPa at 38°C). 595 milk metabolites were analyzed using an untargeted metabolomic analysis. Both treatments differentially altered several classes of compounds. The major changes noted included decreased levels of free fatty acids, phospholipid metabolites, and sphingomyelins. Decreases were more strongly noted in HP samples rather than in HoP ones. Both HoP and HP treatments increased the levels of ceramides and nucleotide compounds. The sterilization of human milk altered its metabolome especially for lipids.

Untargeted metabolomics analysis on kidney tissues from mice reveals potential hypoxia biomarkers.

Chronic hypoxia may have a huge impact on the cardiovascular and renal systems. Advancements in microscopy, metabolomics, and bioinformatics provide opportunities to identify new biomarkers. In this study, we aimed at elucidating the metabolic alterations in kidney tissues induced by chronic hypoxia using untargeted metabolomic analyses. Reverse phase ultrahigh performance liquid chromatography-mass spectroscopy/mass spectroscopy (RP-UPLC-MS/MS) and hydrophilic interaction liquid chromatography (HILIC)-UPLC-MS/MS methods with positive and negative ion mode electrospray ionization were used for metabolic profiling. The metabolomic profiling revealed an increase in metabolites related to carnitine synthesis and purine metabolism. Additionally, there was a notable increase in bilirubin. Heme, N-acetyl-L-aspartic acid, thyroxine, and 3-beta-Hydroxy-5-cholestenoate were found to be significantly downregulated. 3-beta-Hydroxy-5-cholestenoate was downregulated more significantly in male than female kidneys. Trichome Staining also showed remarkable kidney fibrosis in mice subjected to chronic hypoxia. Our study offers potential intracellular metabolite signatures for hypoxic kidneys.

Evaluating the strengths and challenges of PAX dream makers approach to mental health promotion: perspectives of youth and community members in indigenous communities in Manitoba, Canada.

PAX Good Behaviour Game (PAX-GBG) is an evidence-based approach to co-create a nurturing environment where all children can thrive. This school-based approach was identified as a promising intervention for suicide prevention by First Nations communities in Manitoba, Canada. To enhance this mental health promotion approach, PAX Dream Makers was developed. It is a youth-led addition to PAX-GBG for middle and high school students. This study's aim was to examine, from the communities' perspectives, the influence of PAX Dream Makers on youth as well as its strengths, challenges and suggestions for future improvements. A case study method was conducted using interviews and focus groups with 30 youth and 17 adult mentors and elders. Participants reported that PAX Dream Makers provided support and encouragement to the youth, increased their resilience and provided an opportunity to be positive role models. It strengthened PAX-GBG implementation in schools. Challenges included: adult mentors availability, frequent teacher turn-over and community mental distress. Suggestions expressed were: being mindful of cultural and community contexts, increasing community leadership's understanding of PAX-GBG and better recruitment of mentors and youth. PAX Dream Makers approach was well-received by communities and holds great promise for promoting the well-being of First Nations youth.

Inulin Supplementation Mitigates Gut Dysbiosis and Brain Impairment Induced by Mild Traumatic Brain Injury during Chronic Phase.

Mild traumatic brain injury (mTBI) has been shown to acutely alter the gut microbiome diversity and composition, known as dysbiosis, which can further exacerbate metabolic and vascular changes in the brain in both humans and rodents. However, it remains unknown how mTBI affects the gut microbiome in the chronic phase recovery (past one week post injury). It is also unknown if injury recovery can be improved by mitigating dysbiosis. The goal of the study is to fill the knowledge gap. First, we aim to understand how mTBI alters the gut microbiome through the chronic period of recovery (3 months post injury). In addition, as the gut microbiome can be modulated by diet, we also investigated if prebiotic inulin, a fermentable fiber that promotes growth of beneficial bacteria and metabolites, would mitigate dysbiosis, improve systemic metabolism, and protect brain structural and vascular integrity when administered after 3 months post closed head injury (CHI). We found that CHI given to male mice at 4 months of age induced gut dysbiosis which peaked at 1.5 months post injury, reduced cerebral blood flow (CBF) and altered brain white matter integrity. Interestingly, we also found that Sham mice had transient dysbiosis, which peaked 24 hours after injury and then normalized. After 8 weeks of inulin feeding, CHI mice had increased abundance of beneficial/anti-inflammatory bacteria, reduced abundance of pathogenic bacteria, enriched levels of short-chain fatty acids, and restored CBF in both hippocampi and left thalamus, compared to the CHI-control fed and Sham groups. Using machine learning, we further identified top bacterial species that separate Sham and CHI mice with and without the diet. Our results indicate that there is an injury- and time-dependent dysbiosis between CHI and Sham mice; inulin is effective to mitigate dysbiosis and improve brain injury recovery in the CHI mice. As there are currently no effective treatments for mTBI, the study may have profound implications for developing therapeutics or preventive interventions in the future.

Bacille Calmette-Guérin vaccine reprograms human neonatal lipid metabolism in vivo and in vitro.

Vaccines have generally been developed with limited insight into their molecular impact. While systems vaccinology enables characterization of mechanisms of action, these tools have yet to be applied to infants, who are at high risk of infection and receive the most vaccines. Bacille Calmette-Guérin (BCG) protects infants against disseminated tuberculosis (TB) and TB-unrelated infections via incompletely understood mechanisms. We employ mass-spectrometry-based metabolomics of blood plasma to profile BCG-induced infant responses in Guinea-Bissau in vivo and the US in vitro. BCG-induced lysophosphatidylcholines (LPCs) correlate with both TLR-agonist- and purified protein derivative (PPD, mycobacterial antigen)-induced blood cytokine production in vitro, raising the possibility that LPCs contribute to BCG immunogenicity. Analysis of an independent newborn cohort from The Gambia demonstrates shared vaccine-induced metabolites, such as phospholipids and sphingolipids. BCG-induced changes to the plasma lipidome and LPCs may contribute to its immunogenicity and inform the development of early life vaccines.

Characterising methamphetamine use to inform health and social policies in Manitoba, Canada: a protocol for a retrospective cohort study using linked administrative data.

INTRODUCTION: Rising use of methamphetamine is causing significant public health concern in Canada. The biological and behavioural effects of methamphetamine range from wakefulness, vigour and euphoria to adverse physical health outcomes like myocardial infarction, haemorrhagic stroke, arrhythmia and seizure. It can also cause severe psychological complications such as psychosis. National survey data point to increasing rates of methamphetamine use, as well as increasing ease of access and serious methamphetamine-related harms. There is an urgent need for evidence to address knowledge gaps, provide direction to harm reduction and treatment efforts and inform health and social policies for people using methamphetamine. This protocol describes a study that aims to address this need for evidence. METHODS: The study will use linked, whole population, de-identified administrative data from the Manitoba Population Research Data Repository. The cohort will include individuals in the city of Winnipeg, Manitoba, who came into contact with the health system for reasons related to methamphetamine use from 2013 to 2021 and a comparison group matched on age, sex and geography. We will describe the cohort's sociodemographic characteristics, calculate incidence and prevalence of mental disorders associated with methamphetamine use and examine rates of health and social service use. We will evaluate the use of olanzapine pharmacotherapy in reducing adverse emergency department outcomes. In partnership with Indigenous co-investigators, outcomes will be stratified by First Nations and Métis identity. ETHICS AND DISSEMINATION: The study was approved by the University of Manitoba Health Research Ethics Board, and access datasets have been granted by all data providers. We also received approval from the First Nations Health and Social Secretariat of Manitoba's Health Information Research Governance Committee and the Manitoba Métis Federation. Dissemination will be guided by an 'Evidence 2 Action' group of public rightsholders, service providers and knowledge users who will ensure that the analyses address the critical issues.

The efficiency and fidelity of 8-oxo-guanine bypass by DNA polymerases delta and eta.

A DNA lesion created by oxidative stress is 7,8-dihydro-8-oxo-guanine (8-oxoG). Because 8-oxoG can mispair with adenine during DNA synthesis, it is of interest to understand the efficiency and fidelity of 8-oxoG bypass by DNA polymerases. We quantify bypass parameters for two DNA polymerases implicated in 8-oxoG bypass, Pols delta and eta. Yeast Pol delta and yeast Pol eta both bypass 8-oxoG and misincorporate adenine during bypass. However, yeast Pol eta is 10-fold more efficient than Pol delta, and following bypass Pol eta switches to less processive synthesis, similar to that observed during bypass of a cis-syn thymine-thymine dimer. Moreover, yeast Pol eta is at least 10-fold more accurate than yeast Pol delta during 8-oxoG bypass. These differences are maintained in the presence of the accessory proteins RFC, PCNA and RPA and are consistent with the established role of Pol eta in suppressing ogg1-dependent mutagenesis in yeast. Surprisingly different results are obtained with human and mouse Pol eta. Both mammalian enzymes bypass 8-oxoG efficiently, but they do so less processively, without a switch point and with much lower fidelity than yeast Pol eta. The fact that yeast and mammalian Pol eta have intrinsically different catalytic properties has potential biological implications.

Preferential cis-syn thymine dimer bypass by DNA polymerase eta occurs with biased fidelity.

Human DNA polymerase eta (Pol eta) modulates susceptibility to skin cancer by promoting DNA synthesis past sunlight-induced cyclobutane pyrimidine dimers that escape nucleotide excision repair (NER). Here we have determined the efficiency and fidelity of dimer bypass. We show that Pol eta copies thymine dimers and the flanking bases with higher processivity than it copies undamaged DNA, and then switches to less processive synthesis. This ability of Pol eta to sense the dimer location as synthesis proceeds may facilitate polymerase switching before and after lesion bypass. Pol eta bypasses a dimer with low fidelity and with higher error rates at the 3' thymine than at the 5' thymine. A similar bias is seen with Sulfolobus solfataricus DNA polymerase 4, which forms a Watson-Crick base pair at the 3' thymine of a dimer but a Hoogsteen base pair at the 5' thymine (ref. 3). Ultraviolet-induced mutagenesis is also higher at the 3' base of dipyrimidine sequences. Thus, in normal people and particularly in individuals with NER-defective xeroderma pigmentosum who accumulate dimers, errors made by Pol eta during dimer bypass could contribute to mutagenesis and skin cancer.

Overlap between child protection services and the youth justice system: protocol for a retrospective population-based cohort study using linked administrative data in Manitoba, Canada.

INTRODUCTION: Children who have a history of involvement in child protection services (CPS) are over-represented in the youth and adult criminal justice systems. There are significant health and socioeconomic implications for individuals involved in either or both CPS and the justice system. Understanding the 'overlap' between these two systems would provide insight into the health and social needs of this population. This protocol describes a research programme on the relationship between the child welfare and the youth justice systems, looking specifically at the population involved in both CPS and the youth justice system. We will examine the characteristics associated with involvement in these systems, justice system trajectories of individuals with a history of CPS involvement and early adult outcomes of children involved in both systems. METHODS AND ANALYSIS: Administrative data sets will be linked at the individual level for three cohorts born 1991, 1994 and 1998 in Manitoba, Canada. Involvement in CPS will be categorised as 'placed in out-of-home care', 'received in-home services, but was not placed in care' or 'no involvement'. Involvement in the youth justice system will be examined through contacts with police between ages 12 and 17 that either led to charges or did not proceed. Individual, maternal and neighbourhood characteristics will be examined to identify individuals at greatest risk of involvement in one or both systems. ETHICS AND DISSEMINATION: The study was approved by the University of Manitoba Health Research Ethics Board and permission to access data sets has been granted by all data providers. We also received approval for the study from the First Nations Health and Social Secretariat of Manitoba's Health Information Research Governance Committee and the Manitoba Metis Federation. Strategies to disseminate study results will include engagement of stakeholders and policymakers through meetings and workshops, scientific publications and presentations, and social media.

Latent tuberculosis treatment completion rates from prescription drug administrative data.

OBJECTIVE: In the province of Manitoba, Canada, given that latent tuberculosis infection (LTBI) treatment is provided at no cost to the patient, treatment completion rates should be optimal. The objective of this study was to estimate LTBI treatment completion using prescription drug administrative data and identify patient characteristics associated with completion. METHODS: Prescription drug data (1999-2014) were used to identify individuals dispensed isoniazid (INH) or rifampin (RIF) monotherapy. Treatment completion was defined as being dispensed INH for ≥ 180 days (INH180) or ≥ 270 days (INH270) or RIF for ≥ 120 days (RIF120). Logistic regression models tested socio-demographic and comorbidity characteristics associated with treatment completion. RESULTS: The study cohort comprised 4985 (90.4%) persons dispensed INH and 529 (9.6%) RIF. Overall treatment completion was 60.2% and improved from 43.1% in 1999-2003 to 67.3% in 2009-2014. INH180 showed the highest completion (63.8%) versus INH270 (40.4%) and RIF120 (27.0%). INH180 completion was higher among those aged 0-18 years (68.5%) compared with those aged 19+ (61.0%). Sex, geography, First Nations status, income quintile, and comorbidities were not associated with completion. CONCLUSIONS: Benchmark 80% treatment completion rates were not achieved in Manitoba. Factors associated with non-completion were older age, INH270, and RIF120. Access to shorter LTBI treatments, such as rifapentine/INH, may improve treatment completion.

Dietary inulin alters the gut microbiome, enhances systemic metabolism and reduces neuroinflammation in an APOE4 mouse model.

The apolipoprotein ε4 allele (APOE4) is the strongest genetic risk factor for Alzheimer's disease (AD). APOE4 carriers develop systemic metabolic dysfunction decades before showing AD symptoms. Accumulating evidence shows that the metabolic dysfunction accelerates AD development, including exacerbated amyloid-beta (Aß) retention, neuroinflammation and cognitive decline. Therefore, preserving metabolic function early on may be critical to reducing the risk for AD. Here, we show that inulin increases beneficial microbiota and decreases harmful microbiota in the feces of young, asymptomatic APOE4 transgenic (E4FAD) mice and enhances metabolism in the cecum, periphery and brain, as demonstrated by increases in the levels of SCFAs, tryptophan-derived metabolites, bile acids, glycolytic metabolites and scyllo-inositol. We show that inulin also reduces inflammatory gene expression in the hippocampus. This knowledge can be utilized to design early precision nutrition intervention strategies that use a prebiotic diet to enhance systemic metabolism and may be useful for reducing AD risk in asymptomatic APOE4 carriers.

Mutator alleles of yeast DNA polymerase zeta.

The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol zeta), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol zeta mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol zeta active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol zeta catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol eta was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Delta background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Deltarev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol zeta. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol zetain vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol zetain vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol zeta functions in mammals, where REV3 deletion is lethal.

Specialized mismatch repair function of Glu339 in the Phe-X-Glu motif of yeast Msh6.

The major eukaryotic mismatch repair (MMR) pathway requires Msh2-Msh6, which, like Escherichia coli MutS, binds to and participates in repair of the two most common replication errors, single base-base and single base insertion-deletion mismatches. For both types of mismatches, the side chain of E. coli Glu38 in a conserved Phe-X-Glu motif interacts with a mismatched base. The Ovarepsilon of Glu38 forms a hydrogen bond with either the N7 of purines or the N3 of pyrimidines. We show here that changing E. coli Glu38 to alanine results in nearly complete loss of repair of both single base-base and single base deletion mismatches. In contrast, a yeast strain with alanine replacing homologous Glu339 in Msh6 has nearly normal repair for insertion-deletion and most base-base mismatches, but is defective in repairing base-base mismatches characteristic of oxidative stress, e.g. 8-oxo-G.A mismatches. The results suggest that bacterial MutS and yeast Msh2-Msh6 differ in how they recognize and/or process replication errors involving undamaged bases, and that Glu339 in Msh6 may have a specialized role in repairing mismatches containing oxidized bases.

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.