Exam 2: Lecture 5 Flashcards Preview

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Eukaryotic DNA Polymerases

-there are five
-responsible for generating RNA/DNA primers
-elongate leading and lagging strands
-replicate and repair mitochondrial genome and in DNA repair


Polymerase Alpha

-plays important role in generation of short RNA/DNA primers that are required for replication initiation
-lacks 3'-5' exonuclease/proofreading activity (not required because used only to synthesize very short stretches of DNA)


Polymerase Beta

-required to resynthesize short stretches of DNA during repair process
-lacks proofreading activity (not required because used only to synthesize very sort stretches of DNA)
-base-excision repair


Polymerase Gamma

-used to replicate and repair mitochondrial genome
-tasked with replicating large stretches of DNA so requires proofreading activity


Polymerase Delta (looks like cursive g) and Polymerase Epsilon

-tasked with synthesizing long stretches of DNA (leading and lagging strands) and therefore also require proofreading activity
-essential for viability
-Delta responsible for lagging-strand synthesis DNA repair
-Epsilon responsible for leading-strand synthesis


Eukaryotic DNA Polymerases (Error Prone Polymerases)

-encodes several DNA Polymerases that are prone to make mistakes when they are copying DNA
-can be beneficial in certain circumstances
-in some cases damage done to DNA is so severe that it prevents the canonical DNA polymerases from reading the template strand and synthesizing new strands
-i.e. formation of thymine dimers (in response to UV light) if not corrected by cell canonical DNA polymerase will stall when it gets to this part of genome
-when replication stalls, error prone DNA polymerases recruited to site of damage
-binding pocket of error-prone polymerase can recognize and fit thymine dimers
-some polymerases read it correctly and add two adenine residues to newly synthesized strand and others will ad one adenine and one cytosine residue
-latter not ideal but it's better than having replication stop completely
-only 2% of genome contains protein-coding genes there's 98% chance that AC pair will be located within a part of genome that does not code for protein


Polymerase Mistakes and Consequences

-failure to correct mistakes in synthesis, end result is anomalous base pairing
-if incorrect bases are not excised by DNA then they can become permanently incorporated into DNA of daughter cells
-if these changes occur within regulatory regions (promoters and enhancers) could change expression pattern of gene
-if changes occur within coding exon could change sequence of protein


Mismatch Repair System (What?)

-tasked with following replication machinery during DNA synthesis and correcting mistakes made by DNA polymerase
-bacterial version consists of MutS, MutL and MutH (additional proteins required are UvrD helicase, an exonnuclease, DNA polymerase and DNA ligase.
-imperative for this system to fix distortions
-if distortion made by DNA polymerase it will be permanently incorporated into genome of daughter chromosome


Mismatch Repair System (Process?)

-when incompatible nucleotides are paired they make physical distortion in DNA double helix.
-dimer of MutS proteins is continually scanning genome for mismatched nucleotide pairs
-contact with physical distortion prevents MutS from continuing and induces conformational change within protein itself
-MutS-DNA complex is sufficient to initiate repair of distortion
-MutH generates a single-stranded nick within the newly synthesized strand
-nick is made at the sequence GATC
-four base sequence occurs on average every 256 bases
-nick will be made (on average) roughly 256 bases away from the mismatched nucleotide pair and can be made 5` or 3` of the mismatched pair
-single-strand nick is a signal for the cell to delete a portion of the DNA strand
-in order to do this double helix must be unwound by UvrD helicase
-unwound strand removed by exonuclease which removes DNA from the nick to the distortion
-DNA polymerase fills in created gap and DNA ligase will seal polynucleotide chain via formation of phosphodiester bond


Strand Discrimination by MutH

-in bacteria DNA methylation plays an important role in this process
-in bacterial cells chromosomal DNA is methylated at GATC sequences by an enzyme called Dam methylase
-prior to replication DNA is methylated on both strands
-but during DNA synthesis newly synthesized strand is not immediately methylated therefore for a short period of time daughter duplexes contain methyl groups only on template strands (hemi-methylated DNA)
-MutH recognizes and nicks the non-methylated strand
-Eukaryotic DNA is not methylated to extent seen in bacteral systems so strand discrimination occurs via different mechanisms


Comparisons of Mismatch Repair System

-has been identified in eukaryotic cells
-similar to situation in prokaryotes, distortions in DNA that result from incompatible nucleotide base pairing are detected by homologs or MutS protein
-eukaryotic genomes lack MutH homologs (makes double stranded nick)
-eukaryotic homologs of MutL have acquired a nuclease domain which allows it to substitute for Mut H
-not unheard of in prokaryotic world-few bacterial species have nuclease containing MutL homologs.
-in these situations MutL is recruited to MutS-DNA complex and then makes a cut in newly synthesized DNA strand


Repetitive Sequences

-DNA polymerase has trouble replicating regions of DNA that contain repetitive sequences.
-errors that result often involve looping of either template strand or newly synthesized strand
-if template strand loops out then newly synthesized strand will contain deletion
-if new DNA strand loops out then new strand will be lengthened
-addition or loss of nucleotides can often result in disruption of regulatory region or coding exon and can result in disease


Triplet expansion Diseases

-one most studied examples of looping
-ex: CAG sequence is repeated several times, if transcribed and translated properly this will code for protein that contains several Alanine amino acids.
-if undergoes slippage then later after rounds of replication it is possible for this triplet to expand and for the protein to contain more than the normal number of alanine residues
-results in several disorders like Huntington's disease
-caused by non-CAG type expansions