Chapter06_SSM - 65781_CH06_120_136.qxd 8/1/08 12:57 PM Page...

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Chapter 6: Molecular Biology of DNA Replication and Recombination Chapter Summary Replication is semiconservative in that each parental single strand, which serves as a template strand, is found in one of the double-stranded progeny molecules. Semiconservative replication was first demonstrated in the Meselson–Stahl experiment, in which equilibrium density-gradient centrifugation was used to sepa- rate DNA molecules containing two 15 N-labeled strands, two 14 N-labeled strands, or one of each. DNA molecules of prokaryotes usually have a single origin of replication; eukaryotic DNA molecules usually have many origins. Replication requires many proteins, including a helicase to unwind the helix, a gyrase to relieve the torsional stress farther along, and single-stranded DNA-binding proteins to stabilize the indi- vidual template strands. New-strand DNA synthesis always requires a short RNA fragment that serves as a primer. The RNA primer segment is removed at later stages of replication. Primer elongation and daughter- strand synthesis proceed by DNA polymerase first bringing in a nucleotide triphosphate with the correct base (the Watson–Crick pairing partner of the base in the template strand) and then allowing the free 3 –OH group of the growing strand to attach to the 5 –P group of the incoming nucleotide. In this process the terminal pyrophosphate (P–P) from the nucleotide triphosphate is cleaved and released. Because double- stranded DNA is antiparallel, only one strand (the leading strand) grows in the direction of movement of the replication fork. The other strand (the lagging strand) is synthesized in the opposite direction as short fragments (Okazaki fragments) that are subsequently joined together. The base sequence of a DNA molecule can be determined by the incorporation of dideoxynucleotides, which immediately terminate any DNA strand into which they are incorporated, or by monitoring the release of pyrophosphate as each new nucleotide is incorporated into the growing strand. Usually, the DNA molecules of interest are fragmented and complementary strands of each fragment are sequenced. In the assembly phase of sequencing, the sequences of overlapping fragments are combined to yield the complete sequence. Genetic recombination results from breakage and rejoining of DNA strands. Molecular models of recombi- nation differ in the types of DNA breaks that initiate the process and in predictions about the types and 120
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121 frequencies of gene-conversion events. In gene conversion, one allele becomes converted into a homolo- gous allele, which is detected by aberrant segregation such as 3 : 1 or 1 : 3 in fungal asci. Gene conversion is the outcome of mismatch repair in heteroduplexes. The double-strand break and repair model is the cur- rently favored molecular model of homologous recombination, largely on the basis of evidence from bud- ding yeast. In this model, recombination is initiated when a broken duplex invades an intact duplex, form-
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Chapter06_SSM - 65781_CH06_120_136.qxd 8/1/08 12:57 PM Page...

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