DNA Replication and Repair PDF

CHAPTER 13 Introduction DNA Replication and Repair Reproduction is a property of all organisms. DNA duplicates by a process called DNA replication....

CHAPTER 13 Introduction DNA Replication and Repair Reproduction is a property of all organisms. DNA duplicates by a process called DNA replication. The DNA replication machinery is also used for DNA repair. 13.1 DNA Replication (1) DNA Replication (2) DNA replication takes Semiconservative Replication place by separation of – DNA replication is called semiconservative the strands of the because half of the parent structure is double helix, and retained in each of the daughter duplexes. synthesis of two – This model of DNA replication took over the daughter strands other tow models previously considered: complementary to the conservative and dispersive. two parental templates. Three alternate schemes of replication Three alternate schemes of replication Three alternate schemes of replication DNA Replication (3) The Messelson and Stahl experiments supported the semiconservative model of replication in bacterial cells. Semiconservative replication was later demonstrated in eukaryotic cells.  Experimental demonstration of semiconservative Experimental demonstration of semiconservative DNA replication in bacteria DNA replication in eukaryotes DNA Replication (4) DNA Replication (5) Replication in Bacterial Cells Replication Forks and Bidirectional – Temperature-sensitive (ts) mutants were Replication used to identify the genes of replication. – Replication starts at the origin site, where a – Replication can be studied using in vitro number of proteins bind to initiate replication. systems reconstituted from purified cellular – Replication proceeds bidirectionally. compounds. – Replication forks are points where a pair of replicating segments come together and join the nonreplicated segments.  Model of a bacterial chromosome undergoing bidirectional replication DNA Replication (6) Unwinding the Duplex and Separating the Strands – Tension is built up as DNA begins the unwinding process, and the DNA becmes positively supercoiled. – DNA gyrase (topoisomerase II) relieves the tension by changing the DNA into negatively supercoiled DNA. The unwinding problem DNA Replication (7) The Properties of DNA Polymerases – DNA polymerase is responsible for synthesizing new DNA strands from a DNA template. – DNA polymerase requires a primer which provides the 3’ hydroxyl terminus on which to add new nucleotides. – Polymerization occurs in the 5’-to-3’ direction. – None of the three DNA polymerases in bacteria can initiate DNA chains.  Templates and nontemplates for DNA polymerase activity DNA Replication (8) Semidiscontinuous Replication – Both daughter strands are synthesized simultaneously. – The leading strand (in the direction of the replication fork movement) is synthesized continuously. – The lagging strand (in the opposite direction of the replication fork movement) is synthesized discontinuously. The incorporation of nucleotides onto the 3’ The two strands of a double helix are synthesized end of a growing strand by DNA polymerase by a different sequence of events  DNA Replication (9) DNA Replication (10) The lagging strand is Primase is an RNA constructed of small polymerase that Okazaki fragments, assembles short which are joined by RNA primers. DNA ligase. These primers are later removed and the gaps are sealed. The role of DNA helicase, SSB proteins, and DNA Replication (11) primase at the replication fork The Machinery Operating at the Replication Fork – Helicase and single-stranded DNA-binding (SSB) proteins unwind the parental duplex and separate the two strands. – Primase and helicase form a “primosome”, which processively moves along the lagging- strand template. – A single replisome synthesizes both leading and lagging strands. The role of DNA helicase, SSB proteins, and primase at the replication fork DNA Replication (12) The Structure and Functions of DNA Polymerases – DNA polymerase III is the primary replication enzyme. DNA polymerase III holoenzyme contains various subunits having different functions in the replication process. By forming a β clamp, one of the components maintains an association between the polymerase and the DNA template. Replication in E. coli by DNA polymerases working together as part of a complex DNA Replication (13) As long as it is attached to a β “sliding clamp”, DNA polymerase can move processively from one nucleotide to the next. The assembly of the β clamp around the DNA requires a clamp loader, which is part of the DNA polymerase III holoenzyme.  Schematic representation of The β sliding clamp and clamp loader DNA polymerase III The exonuclease activities of DNA Replication (14) DNA polymerase I Exonuclease Activities of DNA Polymerases – DNA polymerase I is involved in DNA repair and also removes RNA primers and replaces them with DNA. – Exonucleases degrade nucleic acids by removing 5’ or 3’ terminal nucleotides.  Geometry of proper and DNA Replication (15) mismatched base pairs Ensuring High Fidelity during DNA Replication – The error rate of incorporation of an incorrect nucleotide during DNA replication is the spontaneous mutation rate. – Incorporation of a particular nucleotide onto the end of growing strand depends upon the geometry of the base pair. Activation of the 3’ à 5’ exonuclease activity DNA Replication (16) of DNA polymerase I Ensuring high fidelity (continued) – During proofreading, mismatched bases are excised. – Careful selection of the nucleotide, proofreading, and mismatch repair account for low error rates in replication (about 10–9). – Replication is rapid (~103 nucleotides/sec).  DNA Replication (17) DNA Replication (18) Replication in Eukaryotic Cells Initiation of Replication in Eukaryotic Cells – Replication is eukaryotes is not as well – Eukaryotes replicate their genome in small portions (replicons). understood as replication in bacteria. Some – Initiation of DNA synthesis in a replicon is regulated. advances include: Using mutant yeast cells unable to produce specific gene products for replication. Development of in vitro systems where replication can occur in cellular extracts or mixtures of purified proteins. Replication of a DNA Replication (19) yeast replicon Origins of replication identified in yeast cells are called autonomous replicating sequences (ARS). A multiprotein origin recognition complex (ORC) is assembled at the ARS. Replication in mammalian cells has been more difficult to study.  DNA Replication (20) DNA Replication (21) Restricting Replication to Once Per Cell The Eukaryotic Replication Fork Cycle – Replication activities are similar in eukaryotes – Replication origins pass through different and prokaryotes. states so that they only replicate their DNA – There are several DNA polymerases in once during a cell cycle. eukaryotes. Origin of replication bound by an ORC. – Eukaryotic DNA polymerases elongate in the Proteins called “licensing factors” bind to the origin. 5’-to-3’ direction and require a primer; some Activation factors bind to the chromosomes and have 3’-to-5’ exonuclease activity. induce “licensed” origins to begin replication. Some Proteins Required for Eukaryotic Schematic view of the major components of DNA Replication the eukaryotic replication fork  The involvement of the nuclear matrix DNA Replication (22) in DNA replication Replication and Nuclear Structure – The replication machinery is stationary in the nuclear matrix. – Replication forks are located within sites called replication foci. – The clustering of replication forks may provide a mechanism for coordinating replication of adjacent replicons on individual chromosomes. Demonstration that replication activities are confined to distinct sites DNA Replication (23) Chromatin Structure and Replication – The assembly of DNA into nucleosomes is a rapid event. – Histones remain intact during replication and old and new histones are distributed randomly between the two daughter duplexes. – The assembly of nucleosomes is facilitated by a network of accessory proteins. The distribution of histone core complexes to daughter cells following replication 13.2 DNA Repair (1) DNA repair is essential for cell survival. – DNA is the cell molecule most susceptible to environmental damage. – Ionizing radiation, common chemicals, UV radiation and thermal energy create spontaneous alteration (lesions) in DNA. – Cells have a number of mechanisms to repair genetic damage. A pyrimidine dimer that has formed within a DNA duplex following UV irradiation DNA Repair (2) Nucleotide Excision Repair – Nucleotide excision repair (NER) removes bulky lesions, such as pyrimidine dimers and chemically altered nucleotides. – It consists of two pathways: A transcription-coupled pathway which is the preferential pathway and selectively repairs genes of greatest importance to the cell. A global genomic pathway which is less efficient and corrects DNA strands in the remainder of the genome. Nucleotide excision repair DNA Repair (3) Nucleotide excision repair (continued) – TFIIH is a key component of the repair machinery and is also involved in the initiation for transcription. It links transcription and DNA repair. – A pair of endonucleases cut on both sides of the lesion, and the damaged strand is removed by helicase. – The gap is filled by a DNA polymerase and sealed by DNA ligase. Base excision repair DNA Repair (4) Base Excision Repair – Base excision repair (BER) removes altered nucleotides that produce distortions of the double helix. – DNA glycosylase recognizes the alteration and cleaves the base form the sugar. – DNA glycosylases are specific for a particular type of altered base.  Detecting damaged bases during BER DNA Repair (5) Base excision repair (continued) – DNA glycosylase removes the altered bases. – Once the altered base is removed, an endonuclease cleaves the DNA backbone and a polymerase fills the gap by inserting a nucleotide complementary to the undamaged strand. – The strand is sealed by DNA ligase. DNA Repair (6) DNA Repair (7) Mismatch repair (MMR) is the correction Double-Strand Breakage Repair of mistakes that escape the DNA – Ionizing radiation (X-rays, gamma rays) along polymerase proofreading activity. with some chemicals cause double-strand – Repair enzymes recognize distortions caused breaks (DSBs). by mismatched bases. – DSBs can be repaired by a pathway in – In bacteria, the parental strands are mammalian cells called nonhomologous end recognized from daughter strands by the joining (NHEJ) in which proteins bind to the presence of methylated bases. broken ends and catalyze reaction to rejoin – Several MMR pathways have been identified the broken ends. in eukaryotes.  Repairing DSBs by NHEJ DNA Repair (8) Double-strand breakage repair (continued) – Cells that lack one of the proteins required for NHEJ are very sensitive to ionizing radiation. – Another DSB repair pathway is homologous recombination, and requires a homologous chromosome to serve as a template for repair of the broken strand. – Defects in both repair pathways have been linked to increased cancer susceptibility. The Human Perspective: The Consequences of DNA Repair Deficiencies (1) Xeroderma pigmentosum Xeroderma pigmentosum (XP) patients cannot repair sun-damaged DNA via NER. Some help for XP patients may become available in the form of skin creams that contain DNA repair enzymes. Cockayne syndrome (CS) patients re deficient in the pathway by which DNA that is transcriptionally active is repaired. The Human Perspective: The Consequences of 13.3 Between Replication and DNA Repair Deficiencies (2) Repair Skin cells with optimal levels of repair Sometimes a DNA lesion is not repaired before enzymes are subject to lesions that fail to DNA is replicated by recruiting a special be excised and repaired. polymerase that bypasses lesions. When polymerase η replicates stretches of Skin cancer is not the only disease damaged DNA, DNA synthesis continues. promoted by deficiency or overworked DNA polymerase η engages in translesion DNA repair systems. synthesis (TLS). Some colon cancer cases are due to TLS polymerases accommodate altered mutations in mismatch repair genes. nucleotides that would not fit within a classic polymerase and have no proofreading activity.