Congenital microcephaly

The underlying etiologies of genetic congenital microcephaly are complex and multifactorial. Recently, with the exponential growth in the identification and characterization of novel genetic causes of congenital microcephaly, there has been a consolidation and emergence of certain themes concerning underlying pathomechanisms. These include abnormal mitotic microtubule spindle structure, numerical and structural abnormalities of the centrosome, altered cilia function, impaired DNA repair, DNA Damage Response signaling and DNA replication, along with attenuated cell cycle checkpoint proficiency. Many of these processes are highly interconnected. Interestingly, a defect in a gene whose encoded protein has a canonical function in one of these processes can often have multiple impacts at the cellular level involving several of these pathways. Here, we overview the key pathomechanistic themes underlying profound congenital microcephaly, and emphasize their interconnected nature

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future

LOSS OF BLOOM SYNDROME PROTEIN CAUSES DESTABILIZATION OF GENOMIC ARCHITECTURE AND IS COMPLEMENTED BY ECTOPIC EXPRESSION OF Escherichia coli RecG IN HUMAN CELLS

Genomic instability driven by non-allelic homologous recombination (NAHR) provides a realistic mechanism that could account for the numerous chromosomal abnormalities that are hallmarks of cancer. We recently demonstrated that this type of instability could be assayed by analyzing the copy number variation of the human ribosomal RNA gene clusters (rDNA). Further, we found that gene cluster instability (GCI) was present in greater than 50% of the human cancer samples that were tested. Here, data is presented that confirms this phenomenon in the human GAGE gene cluster of those cancer patients. This adds credence to the hypothesis that NAHR could be a driving force for carcinogenesis. This data is followed by experimental results that demonstrate the same gene cluster instability in cultured cells that are deficient for the human BLM protein. Bloom’s Syndrome (BS) results from a genetic mutation that results in the abolition of BLM protein, one of human RecQ helicase. Studies of Bloom’s Syndrome have reported a 10-fold increase in sister chromatid exchanges during mitosis which has primarily been attributed to dysregulated homologous recombination. BS also has a strong predisposition to a broad spectrum of malignancies. Biochemical studies have determined that the BLM protein works in conjunction with TOPOIIIα and RMI1/RMI2 to function as a Holliday Junction dissolvase that suppress inadvertent crossover formation in mitotic cells. Because of the similarities in their biochemical activities it was suggested that another DNA helicase found in E. coli, the RecG DNA translocase, is the functional analog of BLM. RecG shares no sequence homology with BLM but it can complement both the sister chromatid exchange elevation and the gene- cluster instability phenotype caused by BLM deficiency. This indicates that the physiological function of BLM that is responsible for these phenotypes rests somewhere in the shared biochemical activities of these two proteins. These data taken together give new insights into the physiological mechanism of BLM protein and the use of Bloom’s Syndrome as a model for carcinogenesis

A study of the Bloom's syndrome protein

Bloom's syndrome is a rare autosomal recessive disorder characterised by an early onset of cancer of many types, erythematous lesions on sun-exposed skin, retarded growth, immunodeficiency and sub- or infertility. Cells from Bloom's syndrome patients have replication defects and an abnormally unstable genome manifested in chromosomal breaks and deletions and in an increased mutation rate. Most characteristically, these cells show elevated levels of sister-chromatid exchanges which probably result from homologous recombination events. Since the cells are not hypersensitive to DNA damaging agents, the defect is unlikely to be in one of the common DNA repair pathways. The gene mutated in Bloom's syndrome, BLM, was cloned in 1995 and found to encode a helicase from the RecQ family. This family is named after its E. coli member, RecQ, and includes at least five human genes. Three of these are mutated in inherited disorders; Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome. In my DPhil project, I have investigated the enzymatic properties of the BLM protein. I have purified the protein in recombinant form and shown that it is a DNA-dependent ATPase and an ATP-dependent helicase with 3'-5' polarity. It binds and unwinds a variety of DNA structures, with a preference for tetraplex (G4)-DNA, Holliday junctions (recombination intermediates) and internal DNA bubbles. Furthermore, it is capable of branch migration, an activity distinct from its helicase activity. BLM forms oligomeric rings with fourfold and sixfold symmetry, both in a cell extract and as purified protein. These results, in combination with the cellular phenotype of Bloom's syndrome and with evidence from the analysis of other RecQ homologues in model organisms such as yeast and E. coli, point to a role for BLM in somatic recombination (recombinational repair). Models for this function are discussed in this thesis

Research on prokaryocyte expression and biological activity of the core region of Bloom’s Syndrome protein

To establish an effective approach for inducing expression of the RecQ core of Bloom’s Syndrome protein (BLM642-1290) and assaying its biological activity in vitro, BLM642-1290 recombinant protein was expressed with IPTG at room temperature in Escherichia coli, and then the expressed product was assayed using SDS-PAGE and western blotting. After purification via affinity chromatography, DNA binding activity and unwinding activity of the protein were assessed by fluorescence polarization. Furthermore, the ATPase activity of the protein was also assayed using ultraviolet spectrophotometry based on PiColorLock Gold reagent. An effective expression method was established for BLM protein in E. coli. The obvious bioactivities of the protein were observed in binding to ssDNA or dsDNA, unwinding the dsDNA in the presence of ATP, as well as catalyzing ATP hydrolysis in the presence of ssDNA in vitro. The prokaryocyte expression method of BLM642-1290 was established successfully and the protein with biological activity was obtained from recombinant E.coli. This would be significant to provide a better understanding on BLM protein and facilitate the elucidation of mechanism of pathopoiesia in Bloom’s Syndrome.Keywords: BLM642-1290 protein, induced expression, enzymatic activit

The RecQ gene family in plants

structure and function. They are 30–50 DNA helicases resolving different recombinogenic DNA structures. The RecQ helicases are key factors in a number of DNA repair and recombination pathways involved in the maintenance of genome integrity. In eukaryotes the number of RecQ genes and the structure of RecQ proteins vary strongly between organisms. Therefore, they have been named RecQ-like genes. Knockouts of several RecQ-like genes cause severe diseases in animals or harmful cellular phenotypes in yeast. Until now the largest number of RecQ-like genes per organism has been found in plants. Arabidopsis and rice possess seven different RecQ-like genes each. In the almost completely sequenced genome of the moss Physcomitrella patens at least five RecQ-like genes are present. One of the major present and future research aims is to define putative plant-specific functions and to assign their roles in DNA repair and recombination pathways in relation to RecQ genes from other eukaryotes. Regarding their intron positions, the structures of six RecQ-like genes of dicots and monocots are virtually identical indicating a conservation over a time scale of 150 million years. In contrast to other eukaryotes one gene (RecQsim) exists exclusively in plants. It possesses an interrupted helicase domain but nevertheless seems to have maintained the RecQ function. Owing to a recent gene duplication besides the AtRecQl4A gene an additional RecQ-like gene (AtRecQl4B) exists in the Brassicaceae only. Genetic studies indicate that a AtRecQl4A knockout results in sensitivity to mutagens as well as an hyperrecombination phenotype. Since AtRecQl4B was still present, both genes must have non-redundant roles. Analysis of plant RecQ-like genes will not only increase the knowledge on DNA repair and recombination, but also on the evolution and radiation of protein families

Role of APC and DNA mismatch repair genes in the development of colorectal cancers

Colorectal cancer is the third most common cause of cancer-related death in both men and women in the western hemisphere. According to the American Cancer Society, an estimated 105,500 new cases of colon cancer with 57,100 deaths will occur in the U.S. in 2003, accounting for about 10% of cancer deaths. Among the colon cancer patients, hereditary risk contributes approximately 20%. The main inherited colorectal cancers are the familial adenomatous polyposis (FAP) and the hereditary nonpolyposis colorectal cancers (HNPCC). The FAP and HNPCC are caused due to mutations in the adenomatous polyposis coli (APC) and DNA mismatch repair (MMR) genes. The focus of this review is to summarize the functions of APC and MMR gene products in the development of colorectal cancers

Cloning And Biochemical Characterization Of The RecQ Helicase And Topoisomerase III In Schizosaccharomyces Pombe

RecQ and Topoisomerase III proteins are both essential for proper chromosomal maintenance in species ranging from bacteria to humans. Due to their highly conserved nature throughout evolution, understanding their basic biochemical properties as well as their interactions together is important for understanding human health, disease, and even to provide potential evolutionary insight. S. pombe has been chosen as a model organism because little research has been done on the biochemical analysis of its RecQ homolog, Rqhl. This study shows developments in the biochemical characterization of a truncation protein of Rqh1 including AFM (Atomic Force Microscopy) Imaging, DNA binding, and ATP hydrolysis activity. New insight of the biochemical properties of Rqh1 will advance the development of a molecular model for Bloom\u27s syndrome and provide a better understanding of the molecular aspects of other diseases associated with mutations m RecQ homologs

Human premature aging, DNA repair and RecQ helicases

Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects