Les génomes de bactériophages ont une capacité remarquable d’évolution. L’objectif de ma thèse a été d’étudier le rôle des recombinases de bactériophages dans cette évolution. Notre hypothèse était que les recombinases phagiques diffèrent de la recombinase bactérienne RecA par une fidélité relâchée lors de la réaction de recherche d’homologie, qui pourrait expliquer en partie la très grande plasticité des génomes de bactériophages. Nous avons tout d’abord utilisé une approche bioinformatique basée sur la recherche d’homologies lointaines pour prédire un maximum de gènes de recombinases dans les génomes entièrement séquencés, puis nous avons confirmé l’activité de recombinaison de certaines d’entre elles, par un test d’appariement d’ADN simple brin entre séquences identiques in vivo. Ceci nous a permis de conclure qu’il existait trois superfamilles de recombinases chez les bactériophages, de type Rad52, Rad51/RecA et Gp2.5, présentes dans 42% des 465 génomes analysés. Dans un deuxième temps, nous avons comparé six de ces recombinases à RecA et avons montré que toutes étaient capables de faire de l’appariement simple brin in vivo, contrairement à RecA. Pour deux d’entre elles, Redβ de Lambda (type Rad52) et Sak4 de HK620 (type Rad51/RecA), nous avons observé que l’appariement simple brin continuait à se produire, avec une efficacité diminuée, jusqu’à 13% de divergence entre les séquences. L’appariement d’ADN simple brin est donc une propriété commune aux recombinases de bactériophages qui les distingue de RecA, et semble pouvoir se maintenir pour un niveau élevé de divergence, ce qui soutient l’hypothèse d’une recombinaison homologue différente et plus relâchée dans sa fidélité chez les bactériophages. / Bacteriophage genomes have a remarkable ability to evolve. The aim of my thesis was to study the role of bacteriophage recombinases in this evolution. Our hypothesis was that such recombinases differ from the bacterial RecA recombinase by a relaxed fidelity during homology search, which may partly explain the high plasticity of bacteriophage genomes. We first used a bioinformatics approach based on remote homology search to predict a maximum of recombinase genes in completely sequenced genomes, and confirmed the recombination activity of some of them by a single-strand DNA annealing assay between identical sequences in vivo. This allowed us to conclude that there were three superfamilies of bacteriophage recombinases, Rad52-like, Rad51/RecA-like and Gp2.5-like, which were present in 42% of the 465 genomes analyzed. In a second step, we compared six of these recombinases to RecA and showed that all were able to anneal single-stranded DNA in vivo, in contrast to RecA. For two of them, Redβ of Lambda (Rad52-like) and Sak4 of HK620 (Rad51/RecAlike), we also observed that they were able to anneal non identical (13% of divergence) single-stranded DNA, with a reduced efficiency. We conclude that the single-stranded DNA annealing is a property common to recombinases of bacteriophages, which is absent in RecA, and seems to tolerate diverged sequences. This supports the hypothesis of a different and more relaxed recombination in bacteriophages.

Doctor of Philosophy / Civil Engineering / Robert J. Peterman / A study was conducted to determine the effect of different concrete properties and prestressing steel indentation types on development length and flexural capacity of pretensioned members. Wires and strands commonly used in the manufacturing of prestressed concrete railroad ties worldwide were selected for the study. Thirteen different 5.32-mm-diameter prestressing wire types and six different strands (four, seven-wire strands and two, three-wire strands) were used to cast prisms with a square cross section. The ratio of concrete to prestressed steel in the test prism’s cross section was representable of typical concrete railroad ties. Thus, geometrical and mechanical properties of test prisms were representative of actual ties in the railroad industry. To understand the effect of concrete-release strengths and slumps on development length, all parameters were kept constant in the prisms except concrete-release strength and slump. To manufacture prisms with different release strengths, all four wires/strands were pulled and detensioned gradually when the concrete compressive strength reached 3500 (24.13 MPa), 4500 (31.03 MPa), and 6000 (41.37 MPa) psi. To determine the effect of different slumps on development length, prisms with different slumps of 3 in. (7.6 cm), 6 in. (15.2 cm), and 9 in. (22.9 cm) were manufactured and all other parameters were kept constant in prisms. All prisms were tested in three-point bending at different spans to obtain estimations of development length based on type of reinforcement, concrete-release strength, and concrete slump. Lastly, a design equation was developed based on experimental data for prediction of development length. In the last phase of load tests, cyclic-loading tests were conducted on the prisms manufactured with wires to evaluate the bond performance of wires with different indentation types under cyclic loading. A total of 210 load tests, including 14 cyclic tests, were conducted. The monotonic-load tests revealed a large difference in the development length of pretensioned concrete members manufactured with different wire/strand types and different concrete-release strengths. Also, the cyclic-load tests revealed a significant difference in bond performance of different wire types under cyclic loading compared to monotonic loading.

<p>To ensure genomic integrity, dividing cells implement multiple checkpoint pathways during the course of the cell cycle. In response to DNA damage, cells may either halt the progression of the cycle (cell cycle arrest) or undergo apoptosis. This choice depends on the extent of damage and the cell's capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks relies on the activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors (e.g., Chk2 and p53) to inhibit cell cycle progression. ATM is an S/T-Q directed kinase that is critical for the cellular response to double-stranded DNA breaks. Following DNA damage, ATM is activated and recruited to sites of DNA damage by the MRN protein complex (Mre11-Rad50-Nbs1 proteins) where ATM phosphorylates multiple substrates to trigger a cell cycle arrest. In cancer cells, this regulation may be faulty and cell division may proceed even in the presence of damaged DNA. We show here that the RSK kinase, often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that RSK disrupts the binding of the MRN complex to DSB DNA. RSK can directly phosphorylate the Mre11 protein at Ser 676 both in vitro and in intact cells and can thereby inhibit loading of Mre11 onto DSB DNA. Accordingly, mutation of Ser 676 to Ala can reverse inhibition of the DSB response by RSK. Collectively, these data point to Mre11 as an important locus of RSK-mediated checkpoint inhibition acting upstream of ATM activation.</p><p>The phosphorylation of Mre11 on Ser 676 is antagonized by phosphatases. Here, we screened for phosphatases that target this site and identified PP5 as a candidate. This finding is consistent with the fact that PP5 is required for the ATM-mediated DNA damage response, indicating that PP5 may promote DSB-induced, ATM-dependent DNA damage response by targeting Mre11 upstream of ATM.</p> / Dissertation

O glioblastoma (GBM) é considerado um dos tumores mais agressivos do sistema nervoso central (SNC). Mesmo com o uso de protocolos modernos de tratamento o prognóstico se mantém bastante reservado, sendo que crianças com GBM apresentam uma sobrevida média de 12 a 15 meses. Mecanismos epigenéticos podem interferir no processo de carcinogênese, sendo que a acetilação do DNA pode modular a expressão de genes que atuam no controle do ciclo celular, contribuindo assim para o desenvolvimento e progressão de neoplasias. Estudos clínicos demonstram que inibidores de histonas deacetilases (HDACs), em monoterapia ou combinados a outros agentes antineoplásicos, são clinicamente ativos e bem tolerados no tratamento de uma ampla variedade de tumores. Estes inibidores podem sensibilizar a resposta celular à irradiação ionizante, possibilitando uma redução nas doses-padrão utilizadas, minimizando os efeitos colaterais a curto e longo prazo. A radiação ionizante induz dano no DNA e é geralmente aceito que quebras da dupla-fita (DSBs) é o tipo de lesão mais severa relacionada à sobrevivência celular e preservação da integridade genômica. No presente estudo, avaliamos o potencial efeito radiosensibilizante do PCI-24781, um novo e potente pan-inibidor de HDAC nas linhagens celulares de GBM pediátrico SF188 e KNS42. Foram comparadas as taxas de proliferação celular, clonogenicidade e apoptose das linhagens SF188 e KNS42 com ou sem tratamento com PCI-24781. Também foram comparadas as taxas de clonogenicidade das linhagens SF188 e KNS42 que foram irradiadas com ou sem tratamento prévio com PCI-24781. Adicionalmente, foram avaliados os efeitos do PCI-24781 na expressão de algumas das principais proteínas responsáveis pelo reparo de quebras da dupla-fita ocasionadas pela irradiação. Para os ensaios de proliferação celular foram utilizados os tempo de 24, 48, 72 e 96h, para apoptose, 48h e para capacidade clonogênica sem irradiação o tempo de 48h, em diferentes doses de PCI-24781 (0,25 - 16 M). O inibidor bloqueou significativamente a proliferação celular (p<0,05), induziu morte por apoptose (p<0,05) e reduziu a capacidade na formação de colônias (p<0,001) em ambas as linhagens. No ensaio para avaliação da radiosensibilidade, foram utilizadas as doses do IC30 11 de cada linhagem do ensaio clonogênico seguida de diferentes doses de irradiação. Ambas as linhagens apresentaram uma significativa (p<0,001) diminuição na formação de colônias em todas as doses de irradiação. A linhagem mais resistente à droga, SF188 foi escolhida para estudo do reparo de quebras da dupla-fita ocasionadas pela irradiação. As expressões da proteína Rad51, importante na via de reparo por recombinação homóloga (HR), e das proteínas DNA-PKcs, Ku70 e Ku86, importantes na via de reparo por união terminal não-homóloga (NHEJ) apresentaram uma maior diminuição quando a linhagem irradiada foi previamente tratada com PCI-24781 em comparação à radioterapia exclusiva. Estes achados demonstram que o inibidor de histona PCI-24781 apresenta um importante papel como agente radiosensibilizante, comprometendo o reparo das quebras de dupla-fita em células de GBM pediátrico tratadas com radioterapia. / Glioblastoma (GBM) is considered one of the most aggressive tumors to affect the central nervous system (CNS). Even employing modern treatment protocols the prognosis remains very poor, with children affected by GBM presenting a median survival rate of 12 to 15 months. Epigenetic mechanisms may interfere with the process of tumorigenesis, and DNA acetylation can modulate the expression of genes that contribute in cell cycle control and participate to the development and progression of cancer. Clinical studies demonstrate that histone deacetylase inhibitors (HDACs), alone or in combination with other antineoplastic agents, are clinically active and well tolerated in the treatment of a wide variety of tumors. These inhibitors may sensitize the cellular response to ionizing radiation, enabling the reduction in standard doses of radiation, ultimately minimizing both short and long-term side effects. Ionizing radiation induces DNA damage and it is generally accepted that the double-stranded breaks (DSBs) is the most severe type of injury related to cell survival and preservation of genomic integrity. In the present study, we evaluated the potential radiosensitizer effect of PCI-24781, a novel potent pan-HDAC inhibitor in the pediatric GBM cell lines SF188 and KNS42. We compared the cell proliferation rates, apoptosis of clonogenicity of KNS42 and SF188, with or without treatment with PCI-24781. Moreover, clonogenicity rates were compared between cell lines that were irradiated with or without prior treatment with PCI-24781 Additionally, we evaluated the effects of PCI-24781 in the expression of some of the major proteins responsible for the repair of double-stranded breaks caused by the irradiation. For the cell proliferation assays, the times of 24, 48, 72 and 96 hours were used, for apoptosis, the time of 48h and clonogenic capacity without irradiation, the time of 48h, and different doses of PCI-24781 (0,25 - 16 M). The inhibitor significantly blocked cell proliferation (p<0,05), inducing cell death by apoptosis (p<0,05) and reducing the colony forming ability (p<0,001) of both lineages. In the assays to evaluate the radiosensitivity , the IC30 doses of the clonogenic assays were used for each cell-line after different doses of irradiation. Both lineages showed a significant decrease (p<0,001) in colony formation at all doses of irradiation. The most resistant cell-line to the drug, SF188, was 13 chosen to study the double-strand breaks repair caused by irradiation. The Rad51 protein levels, critical for homologous recombination (HR), and the DNA-PKcs proteins Ku70 and Ku86, important for DNA repair through non-homologous end joining (NHEJ) showed significant decrease in expression when cell-line was treated with PCI-24781 prior to radiotherapy. These data demonstrates that the histone deacetylase inhibitor PCI-24781 plays an important role as a radiosensitizer agent, compromising the repair of double-strand breaks in pediatric GBM cells following irradiation.

The repair of DNA double-strand breaks (DSBs) is required for the survival of all organisms. In bacteria, DNA DSBs can occur during normal housekeeping processes such as DNA replication or by exogenous damage due to chemicals or radiation. DSBs will compromise the integrity of the genome if left un-repaired, and can be fatal to an organism. Repair of DSBs by homologous recombination (HR) replicates missing chromosomal regions before joining of the separated DNA ends. In Escherichia coli the HR repair steps are; pre-synapsis, synapsis and post-synapsis. In the pre-synaptic stage a DSB is processed into a 3′ single-strand overhang, the substrate required for strand invasion in the synapsis stage and the eventual repair of the DSB. At present there are three identified pre-synapsis systems involved in recombination in bacteria; represented by the AdnAB, AddAB and the RecBCD protein complexes. Each system functions in a similar manner but differ in the physical composition of the machinery. This project investigated the pre-synaptic system of Bacteroides fragilis NCTC9343. Genes encoding putative pre-synapsis proteins were initially identified through analysis of the NCTC9343 genome. The function of these proteins was investigated in vivo by rescue of a repair-deficient strain of E. coli. This demonstrated that Bacteroides fragilis encodes a two component system, where both genes products are required to work in concert for pre-synaptic processing of DSBs. The identified genes were BF2192 and BF2191, and have been renamed addA and addB, respectively. To further examine the role of the AddAB proteins in DSB repair, a Bacteroides fragilis strain with a deletion of addAB was constructed and shown to be extremely sensitive to DNA damaging agents. The AddAB complex was purified and found to be an ATP-dependant helicase and exonuclease that acted on double-stranded DNA ends. In conclusion, this project has identified the proteins involved in pre-synaptic processing of DSBs in B. fragilis NCTC9343, consisting of AddAB homologues, and shown their protective role in repair of DNA damage.

O glioblastoma (GBM) é considerado um dos tumores mais agressivos do sistema nervoso central (SNC). Mesmo com o uso de protocolos modernos de tratamento o prognóstico se mantém bastante reservado, sendo que crianças com GBM apresentam uma sobrevida média de 12 a 15 meses. Mecanismos epigenéticos podem interferir no processo de carcinogênese, sendo que a acetilação do DNA pode modular a expressão de genes que atuam no controle do ciclo celular, contribuindo assim para o desenvolvimento e progressão de neoplasias. Estudos clínicos demonstram que inibidores de histonas deacetilases (HDACs), em monoterapia ou combinados a outros agentes antineoplásicos, são clinicamente ativos e bem tolerados no tratamento de uma ampla variedade de tumores. Estes inibidores podem sensibilizar a resposta celular à irradiação ionizante, possibilitando uma redução nas doses-padrão utilizadas, minimizando os efeitos colaterais a curto e longo prazo. A radiação ionizante induz dano no DNA e é geralmente aceito que quebras da dupla-fita (DSBs) é o tipo de lesão mais severa relacionada à sobrevivência celular e preservação da integridade genômica. No presente estudo, avaliamos o potencial efeito radiosensibilizante do PCI-24781, um novo e potente pan-inibidor de HDAC nas linhagens celulares de GBM pediátrico SF188 e KNS42. Foram comparadas as taxas de proliferação celular, clonogenicidade e apoptose das linhagens SF188 e KNS42 com ou sem tratamento com PCI-24781. Também foram comparadas as taxas de clonogenicidade das linhagens SF188 e KNS42 que foram irradiadas com ou sem tratamento prévio com PCI-24781. Adicionalmente, foram avaliados os efeitos do PCI-24781 na expressão de algumas das principais proteínas responsáveis pelo reparo de quebras da dupla-fita ocasionadas pela irradiação. Para os ensaios de proliferação celular foram utilizados os tempo de 24, 48, 72 e 96h, para apoptose, 48h e para capacidade clonogênica sem irradiação o tempo de 48h, em diferentes doses de PCI-24781 (0,25 - 16 M). O inibidor bloqueou significativamente a proliferação celular (p<0,05), induziu morte por apoptose (p<0,05) e reduziu a capacidade na formação de colônias (p<0,001) em ambas as linhagens. No ensaio para avaliação da radiosensibilidade, foram utilizadas as doses do IC30 11 de cada linhagem do ensaio clonogênico seguida de diferentes doses de irradiação. Ambas as linhagens apresentaram uma significativa (p<0,001) diminuição na formação de colônias em todas as doses de irradiação. A linhagem mais resistente à droga, SF188 foi escolhida para estudo do reparo de quebras da dupla-fita ocasionadas pela irradiação. As expressões da proteína Rad51, importante na via de reparo por recombinação homóloga (HR), e das proteínas DNA-PKcs, Ku70 e Ku86, importantes na via de reparo por união terminal não-homóloga (NHEJ) apresentaram uma maior diminuição quando a linhagem irradiada foi previamente tratada com PCI-24781 em comparação à radioterapia exclusiva. Estes achados demonstram que o inibidor de histona PCI-24781 apresenta um importante papel como agente radiosensibilizante, comprometendo o reparo das quebras de dupla-fita em células de GBM pediátrico tratadas com radioterapia. / Glioblastoma (GBM) is considered one of the most aggressive tumors to affect the central nervous system (CNS). Even employing modern treatment protocols the prognosis remains very poor, with children affected by GBM presenting a median survival rate of 12 to 15 months. Epigenetic mechanisms may interfere with the process of tumorigenesis, and DNA acetylation can modulate the expression of genes that contribute in cell cycle control and participate to the development and progression of cancer. Clinical studies demonstrate that histone deacetylase inhibitors (HDACs), alone or in combination with other antineoplastic agents, are clinically active and well tolerated in the treatment of a wide variety of tumors. These inhibitors may sensitize the cellular response to ionizing radiation, enabling the reduction in standard doses of radiation, ultimately minimizing both short and long-term side effects. Ionizing radiation induces DNA damage and it is generally accepted that the double-stranded breaks (DSBs) is the most severe type of injury related to cell survival and preservation of genomic integrity. In the present study, we evaluated the potential radiosensitizer effect of PCI-24781, a novel potent pan-HDAC inhibitor in the pediatric GBM cell lines SF188 and KNS42. We compared the cell proliferation rates, apoptosis of clonogenicity of KNS42 and SF188, with or without treatment with PCI-24781. Moreover, clonogenicity rates were compared between cell lines that were irradiated with or without prior treatment with PCI-24781 Additionally, we evaluated the effects of PCI-24781 in the expression of some of the major proteins responsible for the repair of double-stranded breaks caused by the irradiation. For the cell proliferation assays, the times of 24, 48, 72 and 96 hours were used, for apoptosis, the time of 48h and clonogenic capacity without irradiation, the time of 48h, and different doses of PCI-24781 (0,25 - 16 M). The inhibitor significantly blocked cell proliferation (p<0,05), inducing cell death by apoptosis (p<0,05) and reducing the colony forming ability (p<0,001) of both lineages. In the assays to evaluate the radiosensitivity , the IC30 doses of the clonogenic assays were used for each cell-line after different doses of irradiation. Both lineages showed a significant decrease (p<0,001) in colony formation at all doses of irradiation. The most resistant cell-line to the drug, SF188, was 13 chosen to study the double-strand breaks repair caused by irradiation. The Rad51 protein levels, critical for homologous recombination (HR), and the DNA-PKcs proteins Ku70 and Ku86, important for DNA repair through non-homologous end joining (NHEJ) showed significant decrease in expression when cell-line was treated with PCI-24781 prior to radiotherapy. These data demonstrates that the histone deacetylase inhibitor PCI-24781 plays an important role as a radiosensitizer agent, compromising the repair of double-strand breaks in pediatric GBM cells following irradiation.

Efficient and accurate repair of DNA double strand breaks (DSBs) is required to maintain genomic stability in both eukaryotes and prokaryotes. In Escherichia coli, DSBs are repaired by homologous recombination (HR). During this process, DNA synthesis needs to be primed and templated from an intact homologous sequence to restore any information that may have been lost on the broken DNA molecule. Two critical late stages of the pathway are repair DNA synthesis and the processing of Holliday junctions (HJs). However, our knowledge of the detailed mechanisms of these steps is still limited. Our laboratory has developed a system that permits the induction of a site-specific DSB in the bacterial chromosome. This break forms in a replication dependent manner on one of the sister chromosomes, leaving the second sister chromosome intact for repair by HR. Unlike previously available systems, the repairable nature of these breaks has made it possible to physically investigate the different stages of DNA double-strand break repair (DSBR) in a chromosomal context. In this thesis, I have addressed some fundamental questions relating to repair DNA synthesis and processing of HJs by using a combination of mutants defective in specific biochemical reactions and an assay that I have developed to detect repair DNA synthesis, using a polar termination sequence (terB). First, by using terB sites located at different locations around the break point, it was shown that the DnaB-dependent repair forks are established in a coordinated manner, meaning that the collision of the repair forks occurs between two repair DNA synthesis initiation sites. Second, DSBR was shown to require the PriB protein known to transduce the DNA synthesis initiation signal from PriA protein to DnaT. Conversely, the PriC protein (known as an alternative to PriB in some reactions) was not required in this process. PriB was also shown to be required to establish DnaB-dependent repair synthesis using the terB assay. Third, the establishment and termination of repair DNA synthesis by collision of converging repair forks were shown to occur independently of HJ resolution. This conclusion results from the comparison of the viability of single and double mutants, deficient in either the establishment of DNA synthesis, HJ resolution or in both reactions, subjected to DSBs and from the study of the DNA intermediates that accumulated in these mutants as detected by two-dimensional gel electrophoresis. Fourth, the role of RecG protein during DSB repair was investigated. Solexa sequencing analyses showed that recG null mutant cells undergoing DSBs accumulate more DNA around the break point (Mawer and Leach, unpublished data). This phenomenon was further investigated by two different approaches. Using terB sites in different locations around the break point and ChIP-Seq analyses to investigate the distribution of RecA in a recG null mutant demonstrating that the establishment of repair forks depends on the presence of RecG. Further studies using PriA helicase-dead mutant showed that the interplay between RecG and PriA proteins is essential for the establishment of correctly oriented repair forks during DSBR. As a whole, this work provides evidence on the coordinated nature of the establishment and termination of DNA synthesis during DSBR and how this requires a correct interplay between PriA-PriB and RecG. A new adapted model of homologous recombination is presented.

A DNA double-strand break (DSB) is a severe form of DNA damage. In fastgrowing cells, DSBs are commonly repaired by homologous recombination (HR) and in E. coli they are exclusively repaired by this mechanism. Failure to accurately repair DSBs can lead to genomic instability. Characterising the DNA intermediates formed during DSB repair by HR is key to understanding this process. A system for inducing a site-specific DSB in the E. coli chromosome has previously been described (Eykelenboom et al., 2008). Here, this system has been used to determine the nature of the intermediates of the repair. It was shown that in a Rec+ background the repair process is rapid and efficient. By contrast, in a ruvAB mutant, which is defective for the Holliday junction (HJ) migration and cleavage complex, RuvABC, HJs are accumulated on both sides of the breakpoint. Replication forks also accumulate at defined positions from the DSB, indicating that unresolved HJs are a barrier to efficient replication that is associated with the repair. This suggests that the resolution of HJs needs to occur prior to the establishment of DNA synthesis. Despite the accumulation of HJs in a ruvAB mutant, cell survival occurs when DSBs are induced for short periods, suggesting that HJs can be resolved in a RuvAB-independent manner. In contrast, the RecG helicase is essential for survival. In a recG mutant, replication forks but not HJs are detected in the region of DSB repair. In a ruvAB recG mutant, intermediates in this region are lost. These observations are consistent with a role of RecG in the stabilisation and maturation of D-loops and not the resolution of Holliday junctions. Nevertheless, an additional role for RecG in later stages of repair cannot yet be excluded. This work provides a solid framework for the further study of DSB repair in E. coli.

The rate of DNA mutation is known to fluctuate across the genome but the patterns of mutation rate variation and molecular causes are poorly defined. It is important to understand these patterns of mutation as they influence where deleterious mutations are likely to arise and how rapidly sequences are likely to accumulate change between species, a measure often used as a proxy for functional constraint. In this work I investigate the relationship between DNA replication and apparent mutation hotspots adjacent to transcription factor binding sites. In eukaryotes both DNA strands are replicated simultaneously, the leading strand as a continuous stretch and the lagging strand as a series of discrete Okazaki fragments that are subsequently ligated together. Some transcription factors are able to bind the DNA lagging strand during replication and act as a partial barrier to DNA polymerase, resulting in the accumulation of Okazaki fragment junctions adjacent to these sites. I find that mutation rate is correlated genome wide with Okazaki junction frequency, suggesting that Okazaki junction processing may be error-prone. We present a mechanistic hypothesis to explain this locally elevated mutation rate and propose a role for lagging strand replication and its error-prone Pol α tract retention in the formation of these hotspots. I test this hypothesis using Okazaki fragment sequencing data from the yeast Saccharomyces cerevisiae to identify peaks in Okazaki junctions. When these peaks are aligned and orientated, so that the direction of lagging strand replication is uniform, I find a peak in substitution rate immediately downstream of Okazaki junctions, precisely where Pol α tract retention is predicted to occur. Novel binding motifs are identified within the underlying DNA of these junctions that can be assigned to known strong and fast-binding transcription factors, previously implicated in the phasing of nucleosomes, such as Reb1. I show that mutation hotspots adjacent to transcription factor binding sites are a conserved feature of eukaryotic genomes. In the human genome I predict sites of preferential Pol α retention using DNase I hypersensitivity footprint data. We observe that those footprints predicted as germline-specific manifest an elevated mutation signature. I propose that the rapid binding of some transcription factors to DNA following replication is required for nucleosome positioning or other important functions, however this incurs a cost in terms of locally elevated mutation rate adjacent to and within the sequence specific binding site. As a consequence these binding sites are biologically important mutational hotspots whose functional significance has been systematically underestimated by standard measures of sequence constraint.

We studied a paleoseismic trench that was excavated across the Banning strand of the San Andreas Fault by Petra Geosciences (33.9172°, -116.538°). The trench exposed a ~40 m wide fault zone in interbedded alluvial sand gravel, silt and clay deposits. We present the first paleoseismic record for the Banning strand of the southern San Andreas Fault. The most recent event occurred sometime between 730 and 950 cal BP, potentially coincident with rupture of the San Gorgonio Pass thrust. We interpret that five earthquakes have occurred since 3.3-2.5 ka and eight earthquakes have likely occurred since 7.1-5.7 ka. It is possible that additional events may have occurred without being recognized, especially in the deeper section the stratigraphy, which was not fully exposed across the fault zone. We calculate an average recurrence interval of 380 - 640 yrs based on four complete earthquake cycles between earthquakes 1 and 5. The average recurrence interval is thus equivalent to or less than the elapsed time since the most recent event on the Banning strand. The recurrence interval is similar to the San Gorgonio Pass (450-1850 years) but longer than that for the Mission Creek strand (~220 years).