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Transcript
BSC 197 6/7/12 DNA Replication, mutagenesis and repair Replication is the molecular duplication of DNA prior to cell division Replication of DNA is semiconservative Two existing strands are separated and used to synthesize new strand Results in two DNA molecules that each have one old and one new strand. This suggests the steps in DNA replication-strand separation and production of new strand Strand separtation Performed by enzyme called helicase Very similar to topoisomerase II Unwinding begins at a particular site referred to as origin of replication Typically one origin on a bacterial chromosome Many on large linear chromosomes Allows more rapid replication of large chromosomes Two strands want to reanneal so they must be prevented from base pairing Single stranded proteins bind to each strand and prevent reannealing DNA Polymerase is the enzyme that duplicates DNA Uses one strand of DNA as a template to make the complementary strand DNA Polymerase moves along strand 3’-5’ and synthesizes DNA 5’-3’ Polymerase needs a “starting block” from which to begin Needs a short duplex structure DNA-RNA duplex is constructed by primase Assembles complementary RNA to each strand DNA polymerase extends RNA primer with DNA RNA primer is later removed and replaced with DNA Replication is typically bidirectional Two replication forks form Replication bubbles expand Each replication fork has a leading and lagging strand Since DNA Polymerase move 3’-5’ it can move continuously on one strand (leading) and discontinously on the other (lagging). The same strand of DNA will be the leading strand on one replication fork and the lagging strand on the other fork Since lagging strand synthesis is discontinuous it is assembled with multiple, small replicated regions called Okazaki fragments. These are later connected by an enzyme called DNA ligase Forms covalent bonds between adjacent nucleotides. Proofreading In addition to the 5’-3’ polymerase activity DNAP also has 3’-5’ exonuclease ability to remove the last nucleotide added Very much slower than polymerase activity Reason for 5’-3’ polymerase activity 3’-5’ polymerase activity would make removal of last nucleotide difficult Polymerase has ability to recognize a mismatch and remove last base Can continue down strand, but slowly Mutation and DNA Repair Mutation-change in DNA sequence Can happen naturally during replication when mismatch is not recognized 1 in 100,000 bases incorrectly added 99% of these are corrected by DNA Polymerase editing 99.9% of remaining errors are corrected by postreplication repair Final rate is approximately 1 in 10 billion bases Can happen as a result of environmental conditions=induced mutations Mutagen=compound that increase mutation rates Radiation UV, X-rays, radon, smoking Free radicals-oxygen or hydrogen radicals can alter DNA Cleared by anti-oxidants like vitamins B or E Can happen randomly through changes in bases More common than uncorrected polymerase errors Tautomeric shift Proton shift can change base structure Thymine: from keto to enol will bind to Guanine Cytosine: from amino to imino will bind to Adeninie Spacing between bases is not significantly different than in proper pair Depurination Loss of base from nucleotide=apurinic or AP site During replication, DNA Polymerase may either leave gap to fill or randomly insert any nucleotide at that position Deamination Conversion of amino group to keto Cytosine becomes Uracil binds to Adenine Adenine becomes Hypoxanthine and binds to Cytosine Pyrimidine dimers Two adjacent pyrimidines bond to each other Causes a kink in the strand Most common between two thymines UV radiation causes thymine dimmers Types of mutations Point mutation-a change in a single base Base substitutions in which a single base is changed Could have pronounced effect if change occurs in promoter or splice site In Open Reading Frame there are three possible outcomes Silent or degenerate mutation Base change has no effect on amino acid encoded by codon Missense mutation Base change results in a new amino acid encoded by codon Nonsense mutation Base change results in a stop codon Frameshift-insertion or deletion of a single base Causes the reading frame to shift by one base Results in dramatically different amino acid sequence Effects of mutations Many mutations have no observable effect on the organism and are called neutral mutations The accumulation of neutral mutations over time is predictable and their presence can be used to determine how closely related two organisms are Genetic equivalent to carbon dating Detrimental mutations-cell or organism is at a disadvantage due to change Natural selection determines this Beneficial mutation-new or altered ability gives an advantage to organisms or cell Natural selection determines this Other terms Null mutant or loss of function mutant-protein encoded by gene is no longer functional after mutation Gain of function-protein encoded by mutated gene has a new function DNA repair DNA Polymerase exonuclease activity and editing are first line Recognition of mismatches or kinks is accomplished by feeling the double strand structure Repair systems sense space between strands If spacing is altered (lesion) repair is made Post replication repair mechanism Repair mechanism detects lesions or gaps in DNA Uses original complementary strand via recombination Gaps can then be filled This mechanism depends on ability to tell old strands from new strands Following replication, new DNA is methylated at GATC sequences There is a delay in methylation New strand is not methylated for a while A later mechanism removes lesions and surrounding regions and fills gaps Ligase closes connections