DNA polymerase beta (Pol beta) fills single nucleotide gaps during base excision repair. Deficiencies in Pol beta can lead to increased mutagenesis and genomic instability in the cell, resulting in cancer. Our laboratory has previously shown that the I260 M somatic mutation of Pol beta, which was first identified in prostate cancer, has reduced nucleotide discrimination in a sequence context-dependent manner. I260 M incorporates the incorrect G opposite A in this context more readily than WT. To identify the molecular mechanism of the reduced fidelity of I260M, we studied incorporation using single turnover kinetics and the nature and rates of conformational changes using steady-state fluorescence and Forster resonance energy transfer (FRET). Our data indicate that the I260 M mutation affects the fingers region of rat Pol beta by creating a "collapsed" state in both the open (in the absence of nucleotide) and closed (prior to chemistry) states. I260 M is a temperature-sensitive mutator and binds nucleotides tighter than the WT protein, resulting in reduced fidelity compared to the WT. Additionally, we have generated a kinetic model of WT and I260 M using FRET and single turnover data, which demonstrates that I260 M precatalytic conformation changes differ compared to the WT as it is missing a precatalytic noncovalent step. Taken together, these results suggest that the collapsed state of I260 M may decrease its ability for nucleotide discrimination, illustrating the importance of the "fingers closing" conformational change for polymerase fidelity and accurate DNA synthesis.

DNA damage is a fundamental molecular cause of genomic instability. Base excision repair (BER) is one line of defense to minimize the potential mutagenicity and/or toxicity derived from damaged nucleobase lesions. However, BER in the context of chromatin, in which eukaryotic genomic DNA is compacted through a hierarchy of DNA-histone protein interactions, is not fully understood. Here, we investigate the activity of BER enzymes at 27 unique geometric locations in a nucleosome core particle (NCP), which is the minimal unit of packaging in chromatin. The BER enzymes include uracil DNA glycosylase (UDG), AP endonuclease 1 (APE1), DNA polymerase β (Pol β), and DNA ligase IIIα complexed with X-ray repair cross complementing group 1 (LigIIIα/XRCC1). This global analysis of BER reveals that initiation of the repair event by UDG is dictated by the rotational position of the lesion. APE1 has robust activity at locations where repair is initiated whereas the repair event stalls at the Pol β nucleotide incorporation step within the central ∼45 bp of nucleosomal DNA. The final step of the repair, catalyzed by LigIIIα/XRCC1, is achieved only in the entry/exit regions of the NCP when nick sites are transiently exposed by unwrapping from the histones. Kinetic assays further elucidate that the location of the damaged lesion modulates enzymatic activity. Notably, these data indicate that some of the BER enzymes can act at a significant number of locations even in the absence of chromatin remodelers or other cellular factors. These results inform genome wide maps of DNA damage and mutations and contribute to our understanding of mutational hotspots and signatures.

Medical schools should have clear policies for students who are pregnant or parents, write Christina Staroschak and Danielle L Sawyer

DNA polymerase beta (Pol β) is a 39 kD vertebrate polymerase that lacks proofreading ability, yet still maintains a moderate fidelity of DNA synthesis. Pol β is a key enzyme that functions in the base excision repair and non-homologous end joining pathways of DNA repair. Mechanisms of fidelity for Pol β are still being elucidated but are likely to involve dynamic conformational motions of the enzyme upon its binding to DNA and deoxynucleoside triphosphates. Recent studies have linked germline and somatic variants of Pol β with cancer and autoimmunity. These variants induce genomic instability by a number of mechanisms, including error-prone DNA synthesis and accumulation of single nucleotide gaps that lead to replication stress. Here, we review the structure and function of Pol β, and we provide insights into how structural changes in Pol β variants may contribute to genomic instability, mutagenesis, disease, cancer development, and impacts on treatment outcomes.

Deletion of genes encoding ribosomal proteins extends lifespan in yeast. This increases translation of the functionally conserved transcription factor Gcn4, and lifespan extension in these mutants is GCN4-dependent. Gcn4 is also translationally upregulated by uncharged tRNAs, as are its Caenorhabditis elegans and mammalian functional orthologs. Here, we show that cytosolic tRNA synthetase inhibitors upregulate Gcn4 translation and extend yeast lifespan in a Gcn4-dependent manner. This cytosolic tRNA synthetase inhibitor is also able to extend the lifespan of C. elegans in an atf-4-dependent manner. We show that mitochondrial tRNA synthetase inhibitors greatly extend the lifespan of C. elegans, and this depends on atf-4. This suggests that perturbations of both cytosolic and mitochondrial translation may act in part via the same downstream pathway. These findings establish GCN4 orthologs as conserved longevity factors and, as long-lived mice exhibit elevated ATF4, leave open the possibility that tRNA synthetase inhibitors could also extend lifespan in mammals.

Within cells, motor and non-motor microtubule-associated proteins (MAPs) simultaneously converge on the microtubule. How the binding activities of non-motor MAPs are coordinated and how they contribute to the balance and distribution of motor transport is unknown. Here, we examine the relationship between MAP7 and tau owing to their antagonistic roles in vivo. We find that MAP7 and tau compete for binding to microtubules, and determine a mechanism by which MAP7 displaces tau from the lattice. MAP7 promotes kinesin-based transport in vivo and strongly recruits kinesin-1 to the microtubule in vitro, providing evidence for direct enhancement of motor motility by a MAP. Both MAP7 and tau strongly inhibit kinesin-3 and have no effect on cytoplasmic dynein, demonstrating that MAPs differentially control distinct classes of motors. Overall, these results reveal a general principle for how MAP competition dictates access to the microtubule to determine the correct distribution and balance of motor activity.