Fan Chung-sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / List of Tables --- p.vii / List of Figures --- p.viii / List of Abbreviations --- p.x / Table of Contents --- p.xi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Nasopharyngeal Carcinoma --- p.1-1 / Chapter 1.1.1 --- Histology of NPC --- p.1-1 / Chapter 1.1.2 --- Etiological Factors --- p.1-2 / Chapter 1.1.3 --- Genetic Changes in NPC --- p.1-5 / Chapter 1.2 --- Background of Present Study --- p.1-14 / Chapter 1.3 --- Aims of Study --- p.1-15 / Chapter Chapter 2 --- Comparative Genomic Hybridization and Fluorescence In- Situ Hybridization / Chapter 2.1 --- Introduction --- p.2-1 / Chapter 2.2 --- Fluorescence In-Situ Hybridization (FISH) --- p.2-2 / Chapter 2.2.1 --- Principle of FISH --- p.2-2 / Chapter 2.2.2 --- Applications of FISH --- p.2-2 / Chapter 2.2.3 --- Advantages and Limitations --- p.2-5 / Chapter 2.3 --- Comparative Genomic Hybridization (CGH) --- p.2-7 / Chapter 2.3.1 --- Principle of CGH --- p.2-7 / Chapter 2.3.2 --- Applications of CGH --- p.2-8 / Chapter 2.3.3 --- Advantages and Limitations --- p.2-10 / Chapter 2.4 --- Method of CGH --- p.2-13 / Chapter 2.4.1 --- CGH Probe Preparation --- p.2-13 / Chapter 2.4.2 --- CGH Template Preparation --- p.2-21 / Chapter 2.4.3 --- Hybridization --- p.2-23 / Chapter 2.4.4 --- Post-hybridization --- p.2-23 / Chapter 2.4.5 --- Fluorescence Detection --- p.2-24 / Chapter 2.4.6 --- Image Acquisition and Analysis --- p.2-24 / Chapter 2.5 --- Method of Interphase FISH --- p.2-29 / Chapter 2.5.1 --- FISH Probe Preparation --- p.2-29 / Chapter 2.5.2 --- FISH Template Preparation --- p.2-29 / Chapter 2.5.3 --- Hybridization --- p.2-30 / Chapter 2.5.4 --- Post-hybridization --- p.2-30 / Chapter 2.5.5 --- Fluorescence Detection --- p.2-30 / Chapter 2.5.6 --- Scoring of FISH Signals --- p.2-31 / Chapter 2.5.7 --- Threshold Determination --- p.2-31 / Chapter Chapter 3 --- FISH Studies on NPC Biopsies Guided by CGH Information Derived from Cell Lines and Xenografts / Chapter 3.1 --- Introduction --- p.3-1 / Chapter 3.2 --- Materials and Methods --- p.3-3 / Chapter 3.2.1 --- CGH Analysis --- p.3-3 / Chapter 3.2.2 --- Interphase FISH Analysis --- p.3-4 / Chapter 3.2.3 --- Statistical Analysis --- p.3-7 / Chapter 3.3 --- Results / Chapter 3.3.1 --- CGH --- p.3-9 / Chapter 3.3.2 --- Interphase FISH Analysis --- p.3-10 / Chapter 3.3.3 --- Statistical Analysis --- p.3-11 / Chapter 3.4 --- Discussion --- p.3-27 / Chapter 3.4.1 --- CGH --- p.3-27 / Chapter 3.4.2 --- Interphase FISH Analysis --- p.3-31 / Chapter 3.5 --- Summary of This Chapter --- p.3-36 / Chapter Chapter 4 --- CGH Studies on Universally Amplified DNA from Microdissected NPC Biopsies and Interphase FISH Analysis / Chapter 4.1 --- Introduction --- p.4-1 / Chapter 4.2 --- Materials and Methods --- p.4-4 / Chapter 4.2.1 --- CGH on Universally Amplified DNA --- p.4-4 / Chapter 4.2.2 --- Interphase FISH Analysis --- p.4-6 / Chapter 4.2.3 --- Statistical Analysis --- p.4-8 / Chapter 4.3 --- Results --- p.4-9 / Chapter 4.3.1 --- CGH on Universally Amplified DNA --- p.4-9 / Chapter 4.3.2 --- Interphase FISH Analysis --- p.4-10 / Chapter 4.3.3 --- Statistical Analysis --- p.4-11 / Chapter 4.3.4 --- Comparison of CGH and FISH Findings --- p.4-11 / Chapter 4.4 --- Discussions --- p.4-30 / Chapter 4.4.1 --- CGH Findings --- p.4-30 / Chapter 4.4.2 --- Interphase FISH Analysis --- p.4-41 / Chapter 4.4.3 --- Comparison of CGH and FISH Findings --- p.4-43 / Chapter 4.5 --- Summary of This Chapter --- p.4-45 / Chapter Chapter 5 --- Conclusion and Further Studies / Chapter 5.1 --- Conclusion --- p.5-1 / Chapter 5.2 --- Further Studies --- p.5-3 / Chapter 5.2.1 --- Combination of Microdissection --- p.5-3 / Chapter 5.2.2 --- Multicolor Karyotyping --- p.5-3 / Chapter 5.2.3 --- Microarrays --- p.5-4 / References --- p.R-1

Knowledge of the distribution of fitness effects of new mutations (DFE) can enable us to quantify the amount of genetic change between species that is driven by natural selection and contributes to adaptive evolution. The primary focus of this thesis is the study of methods to infer the DFE and the study of adaptive evolution in the house mouse subspecies Mus musculus castaneus. Firstly, I extended previous methodology to model the DFE based on polymorphism data. Methods that have previously been used to infer the DFE from polymorphism data have relied on the assumption of a unimodal distribution. I developed new models that can be used to fit DFEs of arbitrary complexity, and found that multimodality can be detected by these models given enough data. I used these new models to analyse polymorphism data from Drosophila melanogaster and M. m. castaneus, and found evidence for a unimodal DFE for D. melanogaster and a bimodal DFE for M. m. castaneus. Secondly, I investigated the contribution of change in coding and non-coding DNA to evolutionary adaptation. I used a polymorphism dataset of ~80 loci from M. m. castaneus sequenced in 15 individuals to investigate selection in protein-coding genes and putatively regulatory DNA close to these genes. I found that, although protein-coding genes are much more selectively constrained than non-coding DNA, they experience similar rates of adaptive substitution. These results suggest that change in functional non-coding DNA sequences might be as important as protein-coding genes to evolutionary adaptation. Thirdly, I used whole genome data from 10 M. m. castaneus individuals to compare the rate of adaptive substitution in autosomal and X-linked genes. I found that, on average, X-linked genes have a 1.8 times faster rate of adaptive substitution than autosomal genes. I also found that faster-X evolution is more pronounced for male-specific genes. I used previously developed theory to show that these observations can be explained if new advantageous mutations are recessive, with an average dominance coefficient less than or equal to 0.25. These results can help to explain the long-studied phenomenon of the large effect of the X chromosome in speciation.

A gene dosage study was conducted on a rare complete trisomy 22 human fibroblast cell line utilizing three lysosomal enzymes, ∝-iduronidase, ∝-galactosidase B, and arylsulfatase A, whose genes are located on chromosome 22 and two control enzymes, ,β-hexosaminidase A and -- fucosidase, with genes not on chromosome 22. A gene dosage effect was clearly demonstrated for an early passage number of the fibroblasts; however, later passage numbers gave inconclusive results. This study suggests that gene dosage studies must be carefully designed to be conducted only on early, matched passage number cells. ∝-fucosidase gave anomalous results most likely due to pleiotropic effects. The present gene dosage study confirmed the trisomic nature of the cell line studied and suggests that this type of study may be a useful diagnostic tool for small deletions, additions, or unbalanced translocations.

In Drosophila oocytes, chromosomes undergo dynamic reorganisation during the prophase of the first meiotic division. This is essential to prepare chromatin for synapsis, recombination and consequent chromosome segregation. The progression of meiotic prophase I is well described, while the molecular mechanisms and regulation of these dramatic chromosomal reorganisations are not well understood. Histone modifying enzymes are major regulators of chromatin structure, however, our knowledge of their roles in meiotic prophase I is still limited. In this work, I investigated the role of the histone demethylase Kdm5/Lid, which removes one of the trimethyl groups at Lys4 of Histone 3 (H3K4me3). I showed that Kdm5/Lid is important for the assembly of the synaptonemal complex, pairing of homologous centromeres, and the karyosome formation. Additionally, Kdm5/Lid promotes crossing over and therefore ensures accurate chromosome segregation. Although loss of Kdm5/Lid dramatically increased the level of H3K4me3 in oocytes, catalytically inactive Kdm5/Lid rescued the above cytological defects. Thereby, I found that Kdm5/Lid regulates chromatin architecture in meiotic prophase I oocytes independently of its demethylase activity. To further identify the regulators of meiotic chromatin organisation during prophase I, I carried out a small-scale RNAi screen for karyosome defects. I found that depletion of ubiquitin ligase components, SkpA, Cul-3 and Ubc-6, disrupted the karyosome formation and the assembly of the synaptonemal complex. The success of the small-scale screen motivated me to initiate the genome-scale RNAi screen for karyosome defects. I found 40 new genes that, when depleted, strongly impaired karyosome morphology. Further studies are required to confirm and elucidate their role in chromatin organisation in oocytes. Overall, my findings have advanced our understanding of the regulation of chromatin reorganisation during oocyte development. Because of the conservation between Drosophila and human meiosis, this study provides novel insights into the regulation of meiotic progression in human oocytes.

XY-women with conditions such as Androgen Insensitivity Syndrome (AIS) have male sex chromosomes, internal (abdominal) testicular or gonadal streak tissue, and no ovaries or (usually) uterus, but are otherwise female in body form and gender identity/role. Many have no reason to doubt a female sex until they are investigated for failure to menstruate. Using mixed-method (quantitative and qualitative) empirical methodology, the study reveals how XY-women discovered their diagnosis, with an in-depth analysis of the medical and societal discourses that shaped the labels/identities to which they have been subjected or they have assumed. Data was collected by questionnaire from 114 women recruited via a peer support group. The study is interdisciplinary, spanning medicine, psychology, sociology and feminist gender theory. It is informed by a range of theories including patriarchy and medicalisation (including terminology issues), sexual dimorphism, sex versus gender, social construction, abjection, self-surveillance and performativity, and sexual difference and corporeality. Many participants had experienced diagnostic secrecy by doctors, particularly in N. America. Younger participants had benefited from a recent move to truth disclosure. Participants had found the androcentric medical discourse/terminology difficult to reconcile with their female appearance, identity and social role; and did not approve of the degree of medicalisation. Infertility was the greatest personal concern but most thought that possession of a vagina was society's main criterion for womanhood. Most seemed secure in their female gender, although some were aware of a degree of performativity. Knowledge of their male biological attributes seemed problematic for many (especially those with Swyer Syndrome1), with expressions of inauthenticity, fraud or freakishness by some. Participants showed little awareness of gender theory and even the idea of a sex versus gender conceptual split seemed confusing for many, but clearer to those in N. America. The majority seemed to construct a totally female sex, although some entertained the idea of an intersexed sex, particularly those in N. America and those with a lesbian or bisexual orientation. The lesbian/bisexual sub-group, and those with a PAIS diagnosis, also showed the greatest awareness of gender performativity. Advocacy is a key aspect of the project, developing the argument that the androcentric focus of intersex medicine and the poor provision of clinical psychology restricts the opportunities for these patients to explore alternative discourses and non-medical meanings of their diagnosis. 1. But needs clarifying using a larger sample.

by Angela Bik-Yu Hui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 119-133). / Acknowledgements / Table of Contents / List of Tables / List of Figures / Abstract / Chapter CHAPTER 1 --- LITERATURE REVIEW --- p.1 / Chapter I. --- Nasopharyngeal carcinoma --- p.1 / Chapter II. --- Etiology of NPC --- p.3 / Chapter II a. --- Geographical & Environmental factors --- p.3 / Chapter II b. --- Epstein-Barr virus Infection --- p.5 / Chapter II c. --- Genetic Factors --- p.8 / Chapter III. --- Cytogenetic Studies of NPC --- p.10 / Chapter III a. --- Traditional Cytogenetics --- p.10 / Chapter III b. --- Previous cytogenetic findings of NPC --- p.12 / Chapter III.c. --- Fluorescence in-situ hybridization --- p.15 / Chapter III.d. --- The new NPC cell line: Cell-666 --- p.18 / Chapter IV. --- Molecular Genetic Studies in NPC --- p.19 / Chapter IV a. --- Oncogenes --- p.20 / Chapter IV b. --- Tumor suppresser genes (TSGs) --- p.22 / Chapter IV c. --- Loss of Heterozygosity Studies --- p.29 / Chapter IV d. --- LOH on Chromosome 11 --- p.32 / Chapter IV e. --- ATM Gene --- p.35 / Chapter CHAPTER 2 --- OBJECTIVE OF STUDY --- p.38 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.41 / Chapter I： --- Study of loss of heterozygosity on chromosome 11 --- p.41 / Chapter I a. --- Patients and Specimens --- p.41 / Chapter I.b. --- DNA extraction --- p.45 / Chapter I c. --- Microsatellite Polymorphism Analysis --- p.47 / Chapter I d. --- Multiplex PCR analysis --- p.52 / Chapter II. --- Cytogenetic Studies --- p.54 / Chapter II a. --- Culture of cell-666 --- p.54 / Chapter II b. --- Cytogenetic Analysis --- p.56 / Chapter II c. --- Fluorescence in-situ hybridization (FISH) --- p.58 / Chapter II d. --- FISH analysis of other NPC cell lines) --- p.62 / Chapter CHAPTER 4 --- RESULTS --- p.63 / Chapter I: --- Study of loss of heterozygosity on chromosome 11 --- p.63 / Chapter I a. --- LOH analysis --- p.63 / Chapter I b. --- Regions with L OH --- p.73 / Chapter I c. --- Multiplex PCR analysis --- p.79 / Chapter II: --- Cytogenetic Study --- p.83 / Chapter II a. --- Cytogenetic analysis of cell-666 --- p.83 / Chapter II.b. --- Fluorescence in-situ Hybridization (FISH) --- p.91 / Chapter CHAPTER 5 --- DTSCUSSION --- p.102 / Chapter I. --- LOH of Chromosome 11 Studies --- p.102 / Chapter II. --- Comparison with LOH studies of other chromosomes --- p.110 / Chapter III. --- Cytogenetic Studies --- p.113 / REFERENCES --- p.119

Orientador: Izolete Aparecida Thomazini Santos / Banca: Newton Key Hokama / Banca: Michele Janegitv Acorsi Valério / Resumo: A Citogenética humana é o estudo dos cromossomos, sua estrutura e sua herança, aplicado à prática da genética médica. Há quase 50 anos as anomalias cromossômicas, alterações microscopicamente visíveis no número e/ou na estrutura dos cromossomos, podem ser responsáveis por uma série de condições clínicas denominadas aberrações cromossômicas, que constituem uma categoria de doenças genéticas constitucionais ou adquiridas. Diversas alterações foram descritas em doenças genéticas e doenças neoplásicas de origem hematológica, auxiliando no diagnóstico, prognostico, classificação e monitoramento da doença. O objetivo do presente estudo foi padronizar a técnica clássica em citogenética através da avaliação do bandeamento G pela digestão enzimática com tripsina e pela desnaturação com tampão citratado em alta temperatura, auxiliando na visualização do cariótipo. Neste estudo utilizamos amostras de sangue periférico de indivíduos normais. A cultura celular foi realizada pelo método de cultura in situ, seguindo a metodologia de Moorhead et al. modificada (1960). Em todos os casos foram obtidas metáfases adequadas para análise e o índice mitótico foi satisfatório. Os resultados obtidos pelo emprego de diferentes técnicas de bandeamento cromossômico revelaram que o bandeamento G pela ação enzimática da tripsina embora considerado padrão-ouro é mais sensível, trabalhoso e ocorre com um número maior de tentativas devido a variabilidade do tempo para que ocorra a degradação das proteínas, sendo necessário o retrabalho tornando-se ineficiente em amostras com baixo índice mitótico e aumentando consequentemente o custo do exame. Enquanto o bandeamento pela técnica utilizando tampão citratado em alta temperatura é simples, produtivo, ágil e de menor custo / Abstract: Human Cytogenetics is the study of chromosomes, their structure and their inheritance, applied to the practice of medical genetics. For nearly fifty years the chromosomal abnormalities, microscopically visible changes in the number and / or structure of chromosomes, may be responsible for a number of clinical conditions known as chromosomal aberrations, which are a category of constitutional or acquired genetic diseases. Several amendments were described in genetic diseases and neoplastic diseases of hematologic origin, aiding in the diagnosis, prognosis, classification and monitoring of the disease. The aim of this study was to standardize the classical technique in cytogenetics by evaluating the banding G by enzymatic digestion with trypsin and by denaturation with citrate buffer at high temperature, assisting in the karyotype view. In this study we used peripheral blood samples of normal individuals. Cell culture was performed by culturing in situ method, following the method of Moorhead et al. modified (1960). In all cases appropriate metaphases were obtained for analysis and the mitotic index was satisfactory. The results obtained by using different chromosome banding techniques revealed that the G banding by the enzymatic action of the trypsin although considered the gold standard it is more sensitive, cumbersome and it occurs with a larger number of retries due to a variability of the time for degradation of the proteins, requiring rework, this way becoming inefficient in samples with low mitotic index and consequently increasing the cost of the exam. While the banding technique using citrate buffer in high temperature is simple, productive, agile and less expensive / Mestre

Le syndrome d'Aicardi (AiC) est caractérisé par ia triade agénésie du corps calleux, spasmes infantiles et lacunes chorlorétiniennes. Cette triade s'accompagne d'un retard mental souvent sévère. Le syndrome survient chez les filles de façon sporadique, selon un mode d'hérédité dominant lié au chromosome X. Une approche de clonage positionnel n'est donc pas possible puisque aucun cas de transmission familiale n'a été répertorié à ce jour. Une puce génomique spécifique de l'X (résolution théorique de 82 kb) a été utilisée pour cribler le génome de 18 filles AIC à la recherche de variations quantitatives délétères. Aucun variant en nombre de copie (CNV) n'a été impliqué dans la pathologie et nous avons exclu chez les 18 patientes de notre étude les grands réarrangements touchant la totalité du gène FLNA, gène évoqué antérieurement comme candidat fonctionnel. Nous avons alors complété cette stratégie par deux études transcriptomiques. Cette approche vise à sélectionner les gènes dont l'expression diffère entre les filles AIC et des témoins. Initialement à partir d'ARN de 3 lignées cellulaires et d'une puce 22 000 clones (22K) nous avons exclu, a priori, par séquençage 5 gènes candidats: A5MT, M5T4, N5BP1, PLXNB3 et 5YN1. Une deuxième étape a été engagée sur des ARN de prélèvements sanguins de 10 couples fille-mère et une puce 44K afin d'enrichir les données et de pallier à l'influence des lignées cellulaires. Outre la sélection de gènes candidats impliqués dans le syndrome, cette approche est surtout vouée à l'identification des fonctions biologiques dérégulées chez les patientes Aicard!. Les groupements fonctionnels des gènes signatures chez les filles révèlent clairement les effets des facteurs âge, heure de prélèvement, variabilité inter-individuelle. Un groupe de gènes annotés par le terme GO " nucléosome " semble être influencé par le facteur " prise d'antiépileptique ". Un logiciel baptisé ACGR (Approach for Candidate Gene Retrieval) a été conçu et prototypé. Le but est de cribler les bases de données biologiques en incluant des données privées (données des puces transcriptomiques) à la recherche des gènes qui, lorsqu'ils sont mutés donnent un phénotype de syndrome d'Aicardi. Par cette approche, les gènes PLXNB3, MADEGl et 5UV39H3 sont trois gènes candidats pour le Syndrome d'Aicardi. Le séquençage de ces trois gènes s'inscrit dans les perspectives à court terme. Ces approches intégratives reflètent l'évolution de nos concepts de recherche passant de la génétique du retard mental à la génomique du retard mental en tenant compte de la multiplicité des réseaux d'interactions et de régulations. / Aicardi syndrome (AIC) is a severe X-linked dominant neurodevelopmental disorder a!fecting almost exclusively females. Chief features include infantile spasms, corpus caliosal agenesis, and chorioretinal abnormalities. Aicardi syndrome is a sporadic disorder and hypothesized to he caused by heterozygous mutations in an X Iinked-gene but up to now no defined candidate region on the X chromosome has been identified. Positional candidate gene approach is not possible because no familial case were reported. Eighteen Ale patients were analyzed with a full-coverage X chromosomal BAC arrays. No disease-associated Copy Number Variant was identified and we excluded total deletion and duplication of FLNA gene wich had been previously pointed out as a functional candidate. To complete this approach, 2 microarrays studies were performed to compare gene expression between Ale patients and a pool of healthy patients. The first study, on RNA extracted Irom Iymphoblastoid cell lines isolated between 3 AIC patients used 22k oligonucleotide microarray. For the screened patients, no deleterious mutations were found in the 6 selected candidate genes (ASMT, PLXNB3, MST4, SYN1, SSR4, and NSBP1). The second study was performed with 44k microarray, on RNA directly extracted from 10 AIC patients blood samples. Functional clustering analyses revealed the effects of the factors: age, time of blood sampie extraction, and inter-individual gene expression variance. A group of gene annotated by "nucleosome" GO term seemed inlluenced by the factor "use of antiepileptic drugs". In a last strategy, we proposed a knowledge-guided approach for retrieving disease-specific candidate genes named ACGR (Approach for Candidate Gene Retrieval). Knowledge embedded in expert's definitions of candidate gene was expressed as relations between genes and the disease. These definitions were used for guiding-data modelling and are converted into views on the data which ultimately led to retrieval of sets of candidate genes. Thus PLXNB3, MADEGl and SUV39H3 were selected as candidate genes. The perspectives of our work will include sequencing analysis of these genes. These integrative approaches reflect the evolution of our concepts and allow, with the use of biological pathways, the transition between the genetics of mental retardation to the genomics of mental retardation.

SbcCD has been shown to cleave a DNA hairpin formed by a palindromic DNA sequence on the lagging strand template of the E. coli chromosome. This activity was exploited to create a unique system for inducing a single site-specific DNA double-strand break (DSB) once per replication cycle. First, this work shows that the SOS response induced by this DSB is only essential for viability following multiple cycles of cleavage and repair. Next, the SOS-inducible inhibitor of cell division SfiA is shown to be dispensable for survival, despite demonstrating that cleavage of the palindrome causes both an increase in cell size and a delay in nucleoid segregation. A model of the E. coli cell cycle is presented to reconcile the observation that growth under chronic DSB induced conditions has no effect on generation time despite causing an increase in cell size. This system of DSB induction was then coupled with fluorescence markers on both sides of the palindrome to visualise the consequence of the DSB in vivo. Cleavage of the DNA hairpin by SbcCD in a recAmutant was used to selectively degrade the chromosome that replicated the palindrome on the lagging strand of replication, allowing two genetically identical sister chromosomes to be distinguished. This approach was used to show that chromosome segregation in E. coli is not random, but results in the segregation of lagging strand replicated DNA to mid-cell and leading strand replicated DNA to cell poles. Finally, this system for visualising the site of an inducible DSB was optimised for use in various other mutant backgrounds to allow the events of DSB repair to be dissected. This work provides a solid basis for further investigation into the relationship between replication, recombination and chromosome segregation in the model organism E. coli.

To maintain genomic integrity, all cells must accurately duplicate their genetic material in order to provide intact and complete copies to each daughter cell following cell division. Successful inheritance of chromosomal information without changing even a single nucleotide requires accurate and robust DNA replication. This requires that cells tightly control replication initiation from the origin(s), processive elongation of the replisome, and the completion of DNA replication by resolving convergent replication forks ensuring that each sequence is duplicated without alteration. Unlike initiation and elongation, the process by which replication forks converge and are resolved into two discrete, inheritable DNA molecules is not well understood. This process must be remarkably efficient, occurring thousands of times per cell division in human cells, and is likely to be a fundamental step in regulating genome stability in all cells. In this dissertation I address how DNA replication completes in the model system Escherichia coli. To achieve this, I examined candidate mutants for impairments in the completion of DNA replication. By evaluating growth, viability, chromosomal copy number, and plasmid stability I identified a requirement for the proteins RecBCD, ExoI, and SbcCD in the completion reaction. SbcCD and ExoI act before RecBCD in the completion reaction and process the DNA intermediates arising as replication forks converge. These enzymes act in the completion reaction without recombination or RecA, but in the absence of the normal process recombination is required to complete DNA replication via an aberrant pathway that results in genomic instability.