Chapter 11 DNA Replication and Recombination Copyright Copyright 2009 Pearson 2009 Pearson Education, Education, Inc. Inc.
11.1 DNA is reproduced by Semiconservative Replication The complementarity of DNA strands allows each strand to serve as a template for synthesis of the other
Three possible modes of DNA replication are possible
The Meselson-Stahl experiment demonstrated that: DNA replication is semiconservative each new DNA molecule consists of one old strand and one newly synthesized strand
The Taylor-Woods- Hughes experiment demonstrated that DNA replication is semiconservative in eukaryotes.
DNA replication begins at the origin of replication and is bidirectional rather than unidirectional. A replicon is the length of DNA that is replicated following one initiation event at a single origin.
11.2 DNA Synthesis in Bacteria Involves Five Polymerases, as well as Other Enzymes
DNA polymerase catalyzes DNA synthesis and requires a DNA template and all four dntps.
Chain elongation occurs in the 5' to 3' direction by addition of one nucleotide at a time to the 3' end. As the nucleotide is added, the two terminal phosphates are cleaved off.
DNA polymerases I, II, and III can elongate an existing DNA strand but cannot initiate DNA synthesis. All three possess 3' to 5' exonuclease activity. But only DNA polymerase I demonstrates 5' to 3' exonuclease activity.
DNA polymerase III is the enzyme responsible for the 5' to 3' polymerization essential in vivo. Its 3' to 5' exonuclease activity allows proofreading. Polymerase I is believed to be responsible for removing the primer and the synthesis that fills gaps produced during synthesis DNA polymerases I, II, IV, and V are involved in various aspects of repair of damaged DNA.
DNA polymerase III has 10 subunits whose functions are shown.
11.3 Many Complex Tasks Must Be Performed during DNA Replication
There are seven key issues that must be resolved during DNA replication unwinding of the helix reducing increased coiling generated during unwinding synthesis of a primer for initiation discontinuous synthesis of the second strand removal of the RNA primers joining of the gap-filling DNA to the adjacent strand proofreading
DnaA binds to the origin of replication and is responsible for the initial steps in unwinding the helix.
Subsequent binding of DnaB and DnaC further opens and destabilizes the helix. Single-stranded binding proteins (SSBPs) stabilize the open conformation. Helicases require the energy normally supplied by the hydrolysis of ATP to break hydrogen bonds and denature the double helix. Unwinding produces supercoiling that is relieved by DNA gyrase, a member of a larger group of enzymes referred to as DNA topoisomerases.
To elongate a polynucleotide chain, DNA polymerase III requires a primer with a free 3'-hydroxyl group. The enzyme primase synthesizes an RNA primer that provides the free 3'-hydroxyl required by DNA polymerase III.
As the replication fork moves, only one strand can serve as a template for continuous DNA synthesis the leading strand. The opposite lagging strand undergoes discontinuous DNA synthesis. The lagging strand is synthesized as Okazaki fragments, each with an RNA primer.
DNA polymerase I removes the primers on the lagging strand and the fragments are joined by DNA ligase. Both DNA strands are synthesized concurrently by looping the lagging strand to invert the physical but not biological direction of synthesis
The β-subunit clamp prevents the core enzyme from falling off the template during DNA synthesis. Proofreading and error correction are an integral part of DNA replication. All of the DNA polymerases have 3' to 5' exonuclease activity that allows proofreading.
11.4 A Summary of DNA Replication in Prokaryotes DNA synthesis at a single replication fork involves DNA polymerase III, single-stranded binding proteins DNA gyrase, DNA helicase, RNA primers
11.5 Replication in Prokaryotes is Controlled by a Variety of Genes
A number of genes involved in DNA replication have been identified by conditional mutations A temperature-sensitive mutation is an example of a conditional mutation. It may not be expressed at a particular permissive temperature, but when mutant cells are grown at a restrictive temperature, the mutant phenotype is expressed and can be studied.
11.6 Eukaryotic DNA Synthesis Is Similar to Synthesis in Prokaryotes, but More Complex
In eukaryotic cells there is more DNA than prokaryotic cells. the chromosomes are linear. the DNA is complexed with proteins. This makes DNA replication more complex in eukaryotes than in bacteria. Eukaryotic chromosomes contain multiple origins of replication to allow the genome to be replicated in a few hours.
Multiple origins of replication along a eukaryotic chromosome
Yeast autonomously replicating sequences (ARSs) contain an 11-bp consensus sequence flanked by other short sequences involved in efficient initiation. The ARSs are initially bound by a group of proteins to form the origin recognition complex (ORC).
Three DNA polymerases are involved in replication of nuclear DNA. One involves mitochondrial DNA replication. Others are involved in repair processes.
Pol α and δ are the major forms of the enzyme involved in initiation and elongation. Pol α possesses low processivity, a term that reflects the length of DNA that is synthesized by an enzyme before it dissociates from the template. Pol α functions in synthesis of the RNA primers during initiation on the leading and lagging strands. Polymerase switching occurs, and Pol α is replaced by Pol δ for elongation.
11.7 Telomeres Provide Structural Integrity at Chromosome Ends but Are Problematic to Replicate Telomeres at the ends of linear chromosomes consist of long stretches of short repeating sequences and preserve the integrity and stability of chromosomes. Lagging strand synthesis at the end of the chromosome is a problem because once the RNA primer is removed, there is no free 3'-hydroxyl group from which to elongate.
Telomerase directs synthesis of the telomere repeat sequence to fill the gap. This enzyme is a ribonucleoprotein with an RNA that serves as the template for the synthesis of its DNA complement.
11.8 DNA Recombination, Like DNA Replication, Is Directed by Specific Enzymes
Genetic recombination involves endonuclease nicking strand displacement ligation branch migration duplex separation to generate the characteristic Holliday structure
Genetic exchange at equivalent positions along two chromosomes with substantial DNA sequence homology is referred to as general, or homologous, recombination. The RecA protein in E. coli promotes the exchange of reciprocal single-stranded DNA molecules.
Gene Conversion Is a Consequence of DNA Recombination. Gene conversion is characterized by nonreciprocal genetic exchange between two closely linked genes.