Exam 2: Lecture 5 Flashcards
1
Q
Eukaryotic DNA Polymerases
A
- there are five
- responsible for generating RNA/DNA primers
- elongate leading and lagging strands
- replicate and repair mitochondrial genome and in DNA repair
2
Q
Polymerase Alpha
A
- 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)
3
Q
Polymerase Beta
A
- 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
4
Q
Polymerase Gamma
A
- used to replicate and repair mitochondrial genome
- tasked with replicating large stretches of DNA so requires proofreading activity
5
Q
Polymerase Delta (looks like cursive g) and Polymerase Epsilon
A
- 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
6
Q
Eukaryotic DNA Polymerases (Error Prone Polymerases)
A
- 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
7
Q
Polymerase Mistakes and Consequences
A
- 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
8
Q
Mismatch Repair System (What?)
A
- 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
9
Q
Mismatch Repair System (Process?)
A
- 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 3of 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
10
Q
Strand Discrimination by MutH
A
- 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
11
Q
Comparisons of Mismatch Repair System
A
- 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
12
Q
Repetitive Sequences
A
- 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
13
Q
Triplet expansion Diseases
A
- 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
