Therapeutic applications of germline testing for cancer predisposition genes in Asia in the real world.

BACKGROUND: Germline genetic testing is traditionally carried out in patients suspected with hereditary cancer syndrome for enhanced cancer surveillance and/or preventive strategies, but is increasingly carried out for therapeutic indications. MATERIALS AND METHODS: We conducted a retrospective review of patients who underwent germline genetic testing at our centre to determine the prevalence of actionable pathogenic germline variants (PGV) and their clinical utility. RESULTS: From 2000 to 2022, 1154 cancer patients underwent germline testing, with the majority (945/1154) tested with multi-gene panels. Four hundred and eleven (35.6%) patients harboured a PGV and 334 (81%) were clinically actionable. BRCA1/2 accounted for 62.3% of actionable mutations, followed by mismatch repair (18%), and other homologous recombination repair (HRR) genes (19.7%). One hundred and fifty-two germline-positive patients have advanced cancers, and 79 received germline-directed therapies (poly ADP ribose polymerase inhibitors = 75; immunotherapy = 4). Median duration of immunotherapy and poly ADP ribose polymerase were 20.5 months (range 5-40 months) and 8 months (range 1-76 months), respectively. Among BRCA/HRR mutation carriers who received platinum-based chemotherapy, pathological complete response rate in the neoadjuvant setting was 53% (n = 17 breast cancers) and objective response rate was >80% in the advanced setting (n = 71). CONCLUSIONS: One-third of cancer patients tested carried a PGV and ∼80% were clinically actionable. Three-quarters of germline-positive advanced cancer patients received germline-directed therapies in the real world, underscoring the practical utility of germline testing to guide cancer therapeutics.

Volunteer-led online group exercise for community-dwelling older people: a feasibility and acceptability study.

BACKGROUND: Despite the clear benefits of physical activity in healthy ageing, engagement in regular physical activity among community-dwelling older adults remains low, with common barriers including exertional discomfort, concerns with falling, and access difficulties. The recent rise of the use of technology and the internet among older adults presents an opportunity to engage with older people online to promote increased physical activity. This study aims to determine the feasibility and acceptability of training volunteers to deliver online group exercises for older adults attending community social clubs. METHODS: This was a pre-post mixed-methods study. Older adults aged ≥ 65 years attending community social clubs who provided written consent and were not actively participating in exercise classes took part in the feasibility study. Older adults, volunteers, and staff were interviewed to determine the acceptability of the intervention. The intervention was a once weekly volunteer-led online group seated strength exercises using resistance bands. The duration of the intervention was 6 months. The primary outcome measures were the feasibility of the intervention (determined by the number of volunteers recruited, trained, and retained, participant recruitment and intervention adherence) and its acceptability to key stakeholders. Secondary outcome measures included physical activity levels (Community Health Model Activities Programme for Seniors (CHAMPS) questionnaire), modified Barthel Index, Health-related quality of life (EQ-5D-5L), frailty (PRISMA-7) and sarcopenia (SARC-F), at baseline and 6 months. RESULTS: Nineteen volunteers were recruited, 15 (78.9%) completed training and 9 (47.3%) were retained after 1 year (mean age 68 years). Thirty older adults (mean age 77 years, 27 female) participated, attending 54% (IQR 37-67) of exercise sessions. Participants had no significant changes in secondary outcome measures, with a trend towards improvement in physical activity levels (physical activity in minutes per week at baseline was 1770 min, and 1909 min at six months, p = 0.13). Twenty volunteers, older adults, and staff were interviewed and found the intervention acceptable. The seated exercises were perceived as safe, manageable, and enjoyable. CONCLUSIONS: Trained volunteers can safely deliver online group exercise for community-dwelling older adults which was acceptable to older adults, volunteers, and club staff. TRIALS REGISTRATION: NCT04672200.

Providing care for older adults in the Emergency Department: expert clinical recommendations from the European Task Force on Geriatric Emergency Medicine.

PURPOSE: Despite the rapidly expanding knowledge in the field of Geriatric Emergency Medicine in Europe, widespread implementation of change is still lacking. Many opportunities in everyday clinical care are missed to improve care for this susceptible and growing patient group. The aim was to develop expert clinical recommendations on Geriatric Emergency Medicine to be disseminated across Europe. METHODS: A group of multi-disciplinary experts in the field of Geriatric Emergency Medicine in Europe was assembled. Using a modified Delphi procedure, a prioritized list of topics related to Geriatric Emergency Medicine was created. Next, a multi-disciplinary group of nurses, geriatricians and emergency physicians performed a review of recent guidelines and literature to create recommendations. These recommendations were voted upon by a group of experts and placed on visually attractive posters. The expert group identified the following eight subject areas to develop expert recommendations on: Comprehensive Geriatric Assessment in the Emergency Department (ED), age/frailty adjusted risk stratification, delirium and cognitive impairment, medication reviews in the ED for older adults, family involvement, ED environment, silver trauma, end of life care in the acute setting. RESULTS: Eight posters with expert clinical recommendations on the most important topics in Geriatric Emergency Medicine are now available through https://posters.geriemeurope.eu/ . CONCLUSION: Expert clinical recommendations for Geriatric Emergency Medicine may help to improve care for older patients in the Emergency Department and are ready for dissemination across Europe.

Volunteer-led physical activity interventions to improve health outcomes for community-dwelling older people: a systematic review.

BACKGROUND: Physical activity (PA) is important for older people to maintain functional independence and healthy ageing. There is strong evidence to support the benefits of physical activity interventions on the health outcomes of older adults. Nonetheless, innovative approaches are needed to ensure that these interventions are practical and sustainable. AIM: This systematic review explores the effectiveness of volunteer-led PA interventions in improving health outcomes for community-dwelling older people. METHODS: Five databases (MEDLINE, Embase, CINAHL, PEDro, Cochrane library) were systematically searched for studies using trained volunteers to deliver PA interventions for community-dwelling older people aged ≥ 65 years. Meta-analysis was not conducted due to included study heterogeneity. RESULTS: Twelve papers describing eight studies (five papers reported different outcomes from the same study) were included in the review. All eight studies included strength and balance exercises and frequency of PA ranged from weekly to three times a week. Volunteer-led exercises led to improvements in functional status measured using the short physical performance battery, timed up and go test, Barthel Index, single leg stand, step touch test, chair stand test, and functional reach. Frailty status identified by grip strength measurement or the use of long-term care insurance improved with volunteer-led exercises. Interventions led to improvement in fear of falls and maintained or improved the quality of life. The impact on PA levels were mixed. CONCLUSION: Limited evidence suggests that volunteer-led PA interventions that include resistance exercise training, can improve outcomes of community-dwelling older adults including functional status, frailty status, and reduction in fear of falls. More high-quality RCTs are needed to investigate the effects of volunteer-led PA interventions among older people.

Can probiotics, prebiotics and synbiotics improve functional outcomes for older people: a systematic review.

PURPOSE: Research evaluating the effect of probiotics, prebiotics and synbiotics (PPS) on laboratory markers of health (such as immunomodulatory and microbiota changes) is growing but it is unclear whether these markers translate to improved functional outcomes in the older population. This systematic review evaluates the effect of PPS on functional outcomes in older people. METHODS: We conducted a systematic review of the effect of PPS in older adults on functional outcomes (physical strength, frailty, mood and cognition, mortality and receipt of care). Four electronic databases were searched for studies published since year 2000. RESULTS: Eighteen studies (including 15 RCTs) were identified. One of five studies evaluating physical function reported benefit (improved grip strength). Two analyses of one prebiotic RCT assessed frailty by different methods with mixed results. Four studies evaluated mood with no benefit reported. Six studies evaluated cognition: four reported cognitive improvement in participants with pre-existing cognitive impairment receiving probiotics. Seven studies reported mortality as a secondary outcome with a trend to reduction in only one. Five studies reported length of hospital stay but only two peri-operative studies reported shorter stays. CONCLUSION: There is limited evidence that probiotics may improve cognition in older people with pre-existing cognitive impairment but no clear evidence of benefit of PPS on physical function, frailty, mood, length of hospitalisation and mortality. Larger studies with more homogenous interventions, accounting for confounding factors, such as diet, co-morbidities and medications, are required. There is currently inadequate evidence to recommend PPS use to older people in general. PROSPERO REGISTRATION NUMBER: PROSPERO registration number is CRD42020173417. Date of PROSPERO registration: 01/05/20.

Tumor molecular profiling of responders and non-responders following pembrolizumab monotherapy in chemotherapy resistant advanced cervical cancer.

Optimal treatment for advanced cervical cancer after first line chemotherapy remains undefined. Immune checkpoint inhibition with pembrolizumab, a programmed cell death protein 1(PD-1) inhibitor, is under investigation. We analyzed the micro-environmental and molecular genetic profile of tumors from 4 patients with metastatic cervical cancer treated with off-label second-line pembrolizumab in an effort to identify predictive biomarkers. All patients received 2 mg/kg of pembrolizumab, 3-weekly until disease progression. Immunohistochemistry(IHC) for PD-1, PD-L1, CD3 and CD8, as well as next generation sequencing (NGS) for 50 cancer-related genes were performed on tumor samples. All patients tolerated treatment well with no discontinuation of treatment due to toxicity. One patient experienced dramatic and prolonged partial response, and remains stable on pembrolizumab with a progression free survival (PFS) of 21 months at the time of reporting of this series. Three patients experienced disease progression as best response. In the exceptional responder, there was no tumoral expression of PD-L1, however, combined positive score (CPS) for PD-L1 was 1 and we identified somatic mutations in ERBB4(R612W), PIK3CA(E542K) and RB1(E365K). In 2 patients, despite progressive disease defined by RECIST v1.1, symptom stabilization on pembrolizumab was observed. The tumors of both patients had PD-1 expression in ≥1% of stromal lymphocytes. All patients with response or clinical benefit had CPS for PD-L1 ≥ 1. NGS revealed PIK3CA mutations in 3 tumors. Pembrolizumab is a promising therapeutic option in advanced cervical cancer. Further evaluation of biomarkers may guide optimal patient selection.

Physical activity among hospitalised older people: insights from upper and lower limb accelerometry.

BACKGROUND: Few studies have explored the activity levels of hospitalised older people and the intra-daily activity patterns in this group have not been described. AIMS: To describe the quantity and daily pattern of physical activity among hospitalised older people using two accelerometers: the ankle-worn StepWatch Activity Monitor (SAM), and the wrist-worn GENEActiv. METHODS: This cross-sectional observational study was conducted on the acute medical wards for older people in one UK hospital. INCLUSION CRITERIA: participants aged ≥ 70 years, and able to mobilise prior to admission. Participants wore both devices for up to seven consecutive days, or until hospital discharge, whichever was sooner. Intra-daily activity levels were analysed hourly over each 24 h period. RESULTS: 38 participants (mean age 87.8 years, SD 4.8) had their activity levels measured using both devices. The SAM median daily step count was 600 (IQR 240-1427). Intra-daily activity analysis showed two peak periods of ambulatory activity between 9 am-11 am and 6 pm-7 pm. With physical activity defined as ≥ 12 milli-g (GENEActiv), the median time spent above this cut-off point was 4.2 h. 62% of this activity time was only sustained for 1-5 min. Acceptability of both devices was high overall, but the wrist-worn device (96%) was more acceptable to patients than the ankle-worn device (83%). CONCLUSION: Activity levels of these hospitalised older people were very low. Most physical activity was sustained over short periods. The intra-daily pattern of activity is an interesting finding which can help clinicians implement time-specific interventions to address the important issue of sedentary behaviour.

Assessment of Physical Activity of Hospitalised Older Adults: A Systematic Review.

BACKGROUND: The assessment of physical activity levels of hospitalised older people requires accurate and reliable measures. Physical activities that older people in hospital commonly engage in include exercises and walking. Measurement of physical activity levels of older inpatients is essential to evaluate the impact of interventions to improve physical activity levels and to determine associations between physical activity in hospital and other health-related outcome measures. OBJECTIVE: To determine which measures are used to measure physical activity of older people in hospital, and to describe their properties and applications. METHOD: A systematic review of four databases: Medline, Embase, CINAHL and AMED was conducted for papers published from 1996 to 2016. Inclusion criteria were participants aged ≥ 65 years and studies which included measures of physical activity in the acute medical inpatient setting. Studies which specifically assessed the activity levels of surgical patients or patients with neurological conditions such as stroke or brain injury were excluded. All study designs were included in the review. RESULTS: 18 studies were included from 127 articles selected for full review. 15 studies used objective measures to measure the physical activity of older inpatients: 11 studies used accelerometers and four used direct systematic observations. Seven accelerometers were identified including the StepWatch Activity Monitor, activPAL, GENEActiv, Kenz Lifecorder EX, Actiwatch-L, Tractivity and AugmenTech Inc. Pittsburgh accelerometer. Three studies used a subjective measure (interviews with nurses and patients) to classify patients into low, intermediate and high mobility groups. The StepWatch Activity Monitor was reported to be most accurate at step-counting in patients with slow gait speed or altered gait. The activPAL was reported to be highly accurate at classifying postures. CONCLUSION: Physical activity levels of older inpatients can be measured using accelerometers. The accuracy of the accelerometers varies between devices and population-specific validation studies are needed to determine their suitability in measuring physical activity levels of hospitalised older people. Subjective measures are less accurate but can be a practical way of measuring physical activity in a larger group of patients.

Hematopoietic stem cell development: Using the zebrafish to identify extrinsic and intrinsic mechanisms regulating hematopoiesis.

Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic hierarchy, giving rise to each of the blood lineages found throughout the lifetime of the organism. Since the genetic programs regulating HSC development are highly conserved between vertebrate species, experimental studies in zebrafish have not only complemented observations reported in mammals but have also yielded important discoveries that continue to influence our understanding of HSC biology and homeostasis. Here, we summarize findings that have established zebrafish as an important conserved model for the study of hematopoiesis, and describe methods that can be utilized for future investigations of zebrafish HSC biology.

Application of an immune-magnetic cell sorting method for CD138-positive plasma cells in FISH analysis of multiple myeloma.

INTRODUCTION: Interphase fluorescence in situ hybridization (FISH) analysis of multiple myeloma (MM) may indiscriminately count signals of nonplasma cells, thus decreasing specificity and sensitivity. We aimed to evaluate the usefulness of an immune-magnetic sorting method for plasma cells in FISH analysis of MM and define optimal sample preparation conditions. METHODS: Plasma cells were purified using EasySep(®) CD138 Positive Selection Cocktail and Magnetic Nanoparticles (Invitrogen). We compared FISH results with and without plasma cell purification for three sample preparation methods: direct harvest, 24-h culture, and 96-h culture with interleukin-4 in five newly diagnosed MM patients. Archived fixed bone marrow cells of 17 MM patients were also studied. RESULTS: The percentage of abnormal cells identified was significantly higher with plasma cell purification than without purification (median, 88.0%; range, 84.0-100.0%vs. 15.0%, 12.5-29.5%, respectively). The three sample preparation methods showed comparable results. Immune-magnetic sorting also significantly increased the percentage of abnormal cells identified in FISH analysis of archived fixed bone marrow cells (P < 0.001). CONCLUSIONS: Immune-magnetic CD138-positive cell sorting significantly increased the percentage of abnormal cells identified in FISH analysis of MM samples for all sample preparation methods. This method could also be applied for retrospective FISH analysis of archived fixed bone marrow cells.

Pharmacokinetics and pharmacodynamics of docetaxel with or without ketoconazole modulation in chemonaive breast cancer patients.

BACKGROUND: We previously found 70 mg flat-dose docetaxel coadministered with ketoconazole to modulate CYP3A4 to be the maximum tolerated dose that resulted in comparable docetaxel area under the plasma concentration-time curve (AUC) as 75-100 mg/m² docetaxel. PATIENTS AND METHODS: We compared cycle 1 docetaxel pharmacokinetics and pharmacodynamics between ketoconazole-modulated (70 mg flat-dose docetaxel, n = 31) and conventional-dosed docetaxel (75 mg/m², n = 51) in chemonaive breast cancer patients in two sequential phase II studies. RESULTS: Ketoconazole-modulated docetaxel resulted in reduced docetaxel clearance (22.05 ± 8.29 versus 36.52 ± 13.39 l/h, P < 0.001), similar docetaxel AUC (3.93 ± 2.77 versus 3.77 ± 2.70 mg/l·h, P = 0.794) and tumor efficacy (cycle 1 responder 52% versus 55%) and less day 8 neutrophil suppression (1.24 ± 1.02 × 109/l versus 0.47 ± 0.56 × 109/l, P < 0.001), grade 4 neutropenia (32.3% versus 72.0%, P < 0.001) and febrile neutropenia (3.2 versus 23.5%, P = 0.015), compared with conventional-dosed docetaxel. Chinese had the lowest docetaxel clearance, highest AUC and most myelosuppression, followed by Malays and Indians, in response to ketoconazole-modulated docetaxel, while no significant interethnic differences were observed with conventional-dosed docetaxel. CONCLUSIONS: Ketoconazole-modulated docetaxel achieved similar docetaxel AUC and tumor efficacy but reduced neutrophil suppression and febrile neutropenia at ∼40% reduced dose, representing a feasible alternative to conventional-dosed docetaxel. Interethnic differences in CYP3A4 inhibition by ketoconazole exist and are important when evaluating the impact of concomitant medications.

The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-c30): validation of English version in Singapore.

OBJECTIVE: This study aimed to validate the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30, English version 3.0) in Singaporean cancer patients. METHODS: In a cross-sectional study, a heterogeneous sample of cancer patients (n = 57) self-administered a questionnaire containing the QLQ-C30, the Short Form 36 Health Survey (SF-36) and assessing health and sociodemographic status. Construct validity was assessed by testing a priori hypotheses that QLQ-C30 scales would be moderately or strongly correlated with SF-36 scales measuring similar dimensions of health-related quality of life (HRQoL) and that subjects reporting mild symptoms would have better HRQoL scores than those reporting severe symptoms. Internal consistency reliability was assessed using Cronbach's alpha. RESULTS: Strength of Spearman's correlations between the QLQ-C30 and SF-36 scales assessing similar dimensions of HRQoL ranged from 0.35 to 0.67. Subjects with mild symptoms had better scores than those with severe symptoms for all six QLQ-C30 HRQoL scales (p < 0.05 for five scales, Mann-Whitney U tests). Cronbach's alpha ranged from 0.19 for the cognitive functioning scale to 0.91 for the global QoL scale. CONCLUSION: This study provides preliminary evidence for the validity and reliability of the EORTC QLQ-C30 in English-speaking Singaporean cancer patients.

Genetic data for the 13 CODIS STR loci in Singapore Chinese.

Allele frequencies for the 13 CODIS STR loci included in the AmpFISTR Profiler Plus and AmpFISTR Cofiler kits (Applied Biosystems, Foster City, USA) were determined in a sample of 209 unrelated Chinese in Singapore. The combined random match probability for the 13 loci is about 6.6 x10(-15) and the overall probability of excluding paternity is 0.9999899. The results demonstrate that the loci are useful for forensic human identification and parentage testing for the Chinese population in Singapore.

STR data for the 13 CODIS loci in Singapore Malays.

Allele frequencies for the 13 CODIS (Combined DNA Index System, USA) STR loci included in the AmpFISTR Profiler Plus and AmpFISTR Cofiler kits (Applied Biosystems, Foster City, USA) were determined in a sample of 197 unrelated Malays in Singapore.

Genetic data for the 13 CODIS STR loci in Singapore Indians.

Allele frequencies for the 13 CODIS short tandem repeat (STR) loci included in the AmpFISTR Profiler Plus and AmpFISTR Cofiler kits (Applied Biosystems, Foster City, USA) were determined in a sample of 174 unrelated Indians in Singapore.

Differential incorporation and removal of antiviral deoxynucleotides by human DNA polymerase gamma.

Mitochondrial toxicity can result from antiviral nucleotide analog therapy used to control human immunodeficiency virus type 1 infection. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Apparent steady-state K(m) and k(cat) values for insertion of 2',3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZT-TP), 2',3'-dideoxy-CTP (ddCTP), 2',3'-didehydro-TTP (D4T-TP), (-)-2',3'-dideoxy-3'-thiacytidine (3TC-TP), and carbocyclic 2',3'-didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utilized by pol gamma in vitro as efficiently as natural deoxynucleotides, whereas AZT-TP, 3TC-TP, and CBV-TP were only moderate inhibitors of DNA chain elongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV from DNA predicts persistence in vivo following successful incorporation. In contrast, removal of 3'-terminal 3TC residues was 50% as efficient as natural 3' termini. Finally, we observed inhibition of exonuclease activity by concentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termination, persistence of these analogs in DNA and inhibition of exonucleolytic proofreading may also contribute to mitochondrial toxicity.

The mitochondrial p55 accessory subunit of human DNA polymerase gamma enhances DNA binding, promotes processive DNA synthesis, and confers N-ethylmaleimide resistance.

Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a smaller accessory protein variously reported to be 43-54 kDa. Immunoblot analysis of the purified, heterodimeric native human polymerase gamma complex identified the accessory subunit as 55 kDa. We isolated the full-length cDNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli and purified the 55-kDa protein to homogeneity. Recombinant Hp55 forms a high affinity, salt-stable complex with Hp140 during protein affinity chromatography. Immunoprecipitation, gel filtration, and sedimentation analyses revealed a 190-kDa complex indicative of a native heterodimer. Reconstitution of Hp140.Hp55 raises the salt optimum of Hp140, stimulates the polymerase and exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate strength and was a specificity for double-stranded primer-template DNA. However, Hp140.Hp55 has a surprisingly high affinity for DNA, and kinetic analyses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 orders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basis for the salt tolerance and high processivity characteristic of DNA polymerase gamma. Observation of native DNA polymerase gamma both as an Hp140 monomer and as a heterodimer with Hp55 supports the notion that the two forms act in mitochondrial DNA repair and replication. Additionally, association of Hp55 with Hp140 protects the polymerase from inhibition by N-ethylmaleimide.

Effect of DNA polymerases and high mobility group protein 1 on the carrier ligand specificity for translesion synthesis past platinum-DNA adducts.

Translesion synthesis past Pt-DNA adducts can affect both the cytotoxicity and mutagenicity of the platinum adducts. We have shown previously that the extent of replicative bypass in vivo is influenced by the carrier ligand of platinum adducts. The specificity of replicative bypass may be determined by the DNA polymerase complexes that catalyze translesion synthesis past Pt-DNA adducts and/or by DNA damage-recognition proteins that bind to the Pt-DNA adducts and block translesion replication. In the present study, primer extension on DNA templates containing site-specifically placed cisplatin, oxaliplatin, JM216, or chlorodiethylenetriamine-Pt adducts revealed that the eukaryotic DNA polymerases beta, zeta, gamma, and human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) had a similar specificity for translesion synthesis past Pt-DNA adducts (dien >> oxaliplatin >/= cisplatin > JM216). Primer extension assays performed in the presence of high mobility group protein 1 (HMG1), which is known to recognize cisplatin-damaged DNA, revealed that inhibition of translesion synthesis by HMG1 also depended on the carrier ligand of the Pt-DNA adduct (cisplatin > oxaliplatin = JM216 >> dien). These data were consistent with the results of gel-shift experiments showing similar differences in the affinity of HMG1 for DNA modified with the different platinum adducts. Our studies show that both DNA polymerases and damage-recognition proteins can impart specificity to replicative bypass of Pt-DNA adducts. This information may serve as a model for further studies of translesion synthesis.

The accessory subunit of Xenopus laevis mitochondrial DNA polymerase gamma increases processivity of the catalytic subunit of human DNA polymerase gamma and is related to class II aminoacyl-tRNA synthetases.

Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase gamma (pol gamma) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol gamma have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol gamma purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol gamma stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions.