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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