DNA Structure DNA Replication Eukaryotic Chromosome Structure Study Questions DNA Structure, Replication and Eukaryotic Chromatin Structure Overheads DNA Structure, Replication and Eukaryotic Chromatin Structure WWW Links Genetic Topics

DNA Replication DNA replication is semi-conservative, one strand serves as the template for the second strand. Furthermore, DNA replication only occurs at a specific step in the cell cycle. The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle. Stage Activity Duration G1 Growth and increase in cell size 10 hr S DNA synthesis 8 hr G2 Post-DNA synthesis 5 hr M Mitosis 1 hr DNA replication has two requirements that must be met: DNA template Free 3' -OH group Proteins of DNA Replication DNA exists in the nucleus as a condensed, compact structure. To prepare DNA for replication, a series of proteins aid in the unwinding and separation of the double-stranded DNA molecule. These proteins are required because DNA must be single-stranded before replication can proceed. DNA Helicases - These proteins bind to the double stranded DNA and stimulate the separation of the two strands. DNA single-stranded binding proteins - These proteins bind to the DNA as a tetramer and stabilize the single-stranded structure that is generated by the action of the helicases. Replication is 100 times faster when these proteins are attached to the single-stranded DNA. DNA Gyrase - This enzyme catalyzes the formation of negative supercoils that is thought to aid with the unwinding process. In addition to these proteins, several other enzymes are involved in bacterial DNA replication. DNA Polymerase - DNA Polymerase I (Pol I) was the first enzyme discovered with polymerase activity, and it is the best characterized enzyme. Although this was the first enzyme to be discovered that had the required polymerase activities, it is not the primary enzyme involved with bacterial DNA replication. That enzyme is DNA Polymerase III (Pol III). Three activities are associated with DNA polymerase I; 5' to 3' elongation (polymerase activity) 3' to 5' exonuclease (proof-reading activity) 5' to 3' exonuclease (repair activity) The second two activities of DNA Pol I are important for replication, but DNA Polymerase III (Pol III) is the enzyme that performs the 5'-3' polymerase function. Primase - The requirement for a free 3' hydroxyl group is fulfilled by the RNA primers that are synthesized at the initiation sites by these enzymes. DNA Ligase - Nicks occur in the developing molecule because the RNA primer is removed and synthesis proceeds in a discontinuous manner on the lagging strand. The final replication product does not have any nicks because DNA ligase forms a covalent phosphodiester linkage between 3'-hydroxyl and 5'-phosphate groups. A General Model for DNA Replication The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase, DNA helicase and the single-stranded DNA binding proteins. A free 3'OH group is required for replication, but when the two chains separate no group of that nature exists. RNA primers are synthesized, and the free 3'OH of the primer is used to begin replication. The replication fork moves in one direction, but DNA replication only goes in the 5' to 3' direction. This paradox is resolved by the use of Okazaki fragments. These are short, discontinuous replication products that are produced off the lagging strand. This is in comparison to the continuous strand that is made off the leading strand. The final product does not have RNA stretches in it. These are removed by the 5' to 3' exonuclease action of Polymerase I. 5. The final product does not have any gaps in the DNA that result from the removal of the RNA primer. These are filled in by the action of DNA Polymerase I. 6. DNA polymerase does not have the ability to form the final bond. This is done by the enzyme DNA ligase. Genetics of E. coli DNA Replication Mutants are powerful tools to study any biochemical process. But to be useful, the scienctist must be able to maintain mutant in a viable state. This poses a problem for mutants ofa essential processes such as DNA replication. If the mutated gene is re quired for DNA replication, it is obvious that the mutant will not last more than one generation. The use of conditional mutants has helped to solve this problem. Conditional mutants express their mutant phenotype only under restricted conditons. A popular form of conditional mutant is the temperature sensitive mutant. Temperature sensitive mutants only express their mutant phenotype at a temperature the organism normally does not confront. Many of these mutants are expressed at elevate d temperatures. Therefore the mutant will grow normally at the permissive temperature and express the mutant phenotype at the elevetated tempterature. The analysis of temperature-sensitive mutants of E. coli has defined a series of genes and their role in DNA synthesis. The following table list some of the genes and their role in E. coli DNA replication. Gene Function dnaA,I,P Initiation dnaB,C Helicase at oriC dnaE,N,Q,X,Z Subunits of DNA polymerase III dnaG Primase gyrA,B Subunits of gyrase lig Ligase oriC Origin of Replication polA DNA polymerase I polB DNA polymerase II rep Helicase ssb Single-stranded DNA binding proteins Copyright © 1997. Phillip McClean