CHAPTER 13 DNA Replication and Repair Introduction Reproduction

CHAPTER 13 DNA Replication and Repair

Introduction • 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 takes place by separation of the strands of the double helix, and synthesis of two daughter strands complementary to the two parental templates.

DNA Replication (2) • Semiconservative Replication – DNA replication is called semiconservative because half of the parent structure is retained in each of the daughter duplexes. – This model of DNA replication took over the other tow models previously considered: conservative and dispersive.

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 DNA replication in bacteria

Experimental demonstration of semiconservative DNA replication in eukaryotes

DNA Replication (4) • Replication in Bacterial Cells – Temperature-sensitive (ts) mutants were used to identify the genes of replication. – Replication can be studied using in vitro systems reconstituted from purified cellular compounds.

DNA Replication (5) • Replication Forks and Bidirectional Replication – Replication starts at the origin site, where a number of proteins bind to initiate replication. – Replication proceeds bidirectionally. – 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’ end of a growing strand by DNA polymerase

The two strands of a double helix are synthesized by a different sequence of events

DNA Replication (9) • The lagging strand is constructed of small Okazaki fragments, which are joined by DNA ligase.

DNA Replication (10) • Primase is an RNA polymerase that assembles short RNA primers. • These primers are later removed and the gaps are sealed.

DNA Replication (11) • 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 laggingstrand template. – A single replisome synthesizes both leading and lagging strands.

The role of DNA helicase, SSB proteins, and primase at the replication fork

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 DNA polymerase III

The β sliding clamp and clamp loader

DNA Replication (14) • 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.

The exonuclease activities of DNA polymerase I

DNA Replication (15) • 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.

Geometry of proper and mismatched base pairs

DNA Replication (16) • 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).

Activation of the 3’ 5’ exonuclease activity of DNA polymerase I

DNA Replication (17) • Replication in Eukaryotic Cells – Replication is eukaryotes is not as well understood as replication in bacteria. Some 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.

DNA Replication (18) • Initiation of Replication in Eukaryotic Cells – Eukaryotes replicate their genome in small portions (replicons). – Initiation of DNA synthesis in a replicon is regulated.

DNA Replication (19) • 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.

Replication of a yeast replicon

DNA Replication (20) • Restricting Replication to Once Per Cell Cycle – Replication origins pass through different states so that they only replicate their DNA once during a cell cycle. • Origin of replication bound by an ORC. • Proteins called “licensing factors” bind to the origin. • Activation factors bind to the chromosomes and induce “licensed” origins to begin replication.

DNA Replication (21) • The Eukaryotic Replication Fork – Replication activities are similar in eukaryotes and prokaryotes. – There are several DNA polymerases in eukaryotes. – Eukaryotic DNA polymerases elongate in the 5’-to-3’ direction and require a primer; some have 3’-to-5’ exonuclease activity.

Some Proteins Required for Eukaryotic DNA Replication

Schematic view of the major components of the eukaryotic replication fork

DNA Replication (22) • 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.

The involvement of the nuclear matrix in DNA replication

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.

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.

Base excision repair

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) • Mismatch repair (MMR) is the correction of mistakes that escape the DNA polymerase proofreading activity. – Repair enzymes recognize distortions caused by mismatched bases. – In bacteria, the parental strands are recognized from daughter strands by the presence of methylated bases. – Several MMR pathways have been identified in eukaryotes.

DNA Repair (7) • Double-Strand Breakage Repair – Ionizing radiation (X-rays, gamma rays) along with some chemicals cause double-strand breaks (DSBs). – DSBs can be repaired by a pathway in mammalian cells called nonhomologous end joining (NHEJ) in which proteins bind to the broken ends and catalyze reaction to rejoin the broken ends.

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 (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.

Xeroderma pigmentosum

The Human Perspective: The Consequences of DNA Repair Deficiencies (2) • Skin cells with optimal levels of repair enzymes are subject to lesions that fail to be excised and repaired. • Skin cancer is not the only disease promoted by deficiency or overworked DNA repair systems. • Some colon cancer cases are due to mutations in mismatch repair genes.

13. 3 Between Replication and Repair • Sometimes a DNA lesion is not repaired before DNA is replicated by recruiting a special polymerase that bypasses lesions. • When polymerase η replicates stretches of damaged DNA, DNA synthesis continues. • DNA polymerase η engages in translesion synthesis (TLS). • TLS polymerases accommodate altered nucleotides that would not fit within a classic polymerase and have no proofreading activity.