DNA Replication Flashcards
Function of helicase:
unwinds DNA
What is the helicase located in prokaryotes?
DnaB helicase
What is the helicase located in eukaryotes?
MCM protein
Function of topoisomerases:
prevents excessive supercoiling of DNA as helicase unwinds DNA
- measured by linking number
Types of topoisomerases:
- type 1: cuts DNA strand 1 time and corrects linking number by 1
- type 2: cuts DNA strand 2 times and corrects linking number by 2 (DNA gyrase in prok)
Function of DNA gyrase in prok:
introduces negative supercoils for packaging
- type 2 topoisomerase
Function of primase:
produces RNA complement to DNA template strand de novo
- doesn’t need 3’ OH like DNA polymerase
Function of DNA polymerase:
catalyzes the joining of 2 nucleotides by phosphodiester bond
- synthesis only occurs in 5’ to 3’ direction
- needs 3’ OH to start
- can only extend
Function of clamp:
binds to DNA polymerase and helps increase processivity of replication
Name of clamp in prok:
beta clamp
Name of clamp in euk:
PCNA
What is processivity?
the ability to carry out continuous DNA synthesis without dissociation
Function of clamp loader:
necessary to open and assemble clamp onto the DNA strand
- needs E
Function of SSB (single stranded binding proteins):
binds cooperatively to newly separated DNA strand once helicase unwinds DNA
- enhances template exposure
DNA polymerase activities:
- 5’ to 3’ DNA synthesis ONLY
- 5’ to 3’ exonuclease activity
- 3’ to 5’ exonuclease activity
What is 5’ to 3’ exonuclease activity?
- occurs after synthesis occurs
- removes primers and damaged sections of DNA
- repairs DNA
What is 3’ to 5’ exonuclease activity?
- removes nucleotides from 3’ end
- proofreads during synthesis
- increases fidelity of replication
Activities of DNA polymerase I in prok:
- 5’ to 3’ exonuclease activity
- 3’ to 5’ exonuclease activity
- 5’ to 3’ synthesis
Activities of DNA polymerase III in e. coli:
- 3’ to 5’ exonuclease activity
- 5’ to 3’ synthesis
- has a clamp, which increases processivity
How does RNA primer get removed in prok?
5’ exonuclease activity from DNA polymerase 1
How does RNA primer get removed in euk?
RNase H1
What happens if there is a problem with helicase or topoisomerase?
- increases the risk of chromosomal breaks, which leads to chromosomes missing or not joined together correctly
- increases cancer risk
A drug targeting euk or human enzymes will lead to…
a decrease or stop replication in dividing cancer cells
A drug that targets prok enzymes will…
stop bacterial infections by decreases cell division in bacteria
What do Cipro and other quinolone antibiotics target in prok?
topoisomerase type 2
Function of telomerase:
adds nucleotides to 3’ end of DNA
How does telomerase add nucleotides?
- RNA sequence portion of the telomerase is used as template
- extends DNA by complementing RNA sequence of telomerase
- telomerase slides down to new addition of DNA and adds more compliments
Importance of telomerases:
ensures that chromosomes don’t shorten with each replication
Difference between euk and prok chromosomes:
euk: linear and in nucleus
prok: circular and in nucleoid
Function of histones:
packages DNA in a highly condensed form via wrapping
In order to initiate synthesis of DNA, what has to happen with histones?
they have to be removed
What is chromatin?
DNA-binding proteins
- histones
- non-histones
How do histones wrap back onto DNA?
chromatin assembly factors re-package DNA after synthesis is done
What is the end replication problem?
DNA gets shortened after every replication cycle
- only for linear chromosomes
Why does the end replication problem exist?
- RNA primers removed via RNase H1 = only primers on 5’ side of original strand can be replaced by using 3’ OH end of DNA fragment to extend it
- primer on 5’ end of new strand doesn’t have any DNA in front of it so once it leaves, there’s no 3’ OH to extend it
What are telomeres?
DNA sequences that are repeated on the end of linear chromosomes
- gets shortened due to end replication problem and eventually only coding sequences are left
Cells with rapid division will have higher levels of…
telomerase, which will continually extend the telomere
What would the complement strand of this following sequence have?
5’ ATG TGC ACG CAA 3’
3’ TAC ACG TGC GTT 5’
What is the corresponding RNA transcript sequence of the following sequence?
3’ TAC ACG TGC GTT 5’ (anticoding)
5’ AUG UGC ACG CAA 3’
What is the coding strand given the RNA transcript below?
5’ AUG UGC ACG CAA 3’
5’ ATG TGC ACG CAA 3’
- same, just that T and U are different
What is a silent mutation?
- different codon created
- same amino acid
What is a missense mutation?
- different codon created
- incorrect amino acid
What is a nonsense mutation?
- different codon created
- becomes a stop codon
What are some proofreading mechanisms?
- DNA replication proofreading: 3’ exonuclease activity
- transcription proofreading: back tracking
- translation proofreading: EF-Tu protein
What are some DNA errors?
- insertion/deletion
- depurination
- deamination
- tautomeric
- transitions
- transversions
Definition of insertion/deletion DNA errors:
addition/subtraction of one or more nucleotides during copying
- usually in high repeating sequences
Definition of depurination:
removal of purine group by hydrolysis
Definition of deamination:
removal of amine group by hydrolysis
Definition of tautomeric errors:
nitrogenous base has an alternate protonation state, which alters H-bonding between base pairs
Definition of transition errors:
result in mismatch, but still has one purine and one pyrimidine
Definition of transversion errors:
result in mismatch with 2 purines or 2 pyrimidines
During viral replication, the virus has…
long terminal repeats (LTR)
LTRs allow:
- primer reuse and recognition
- cyclization of genome into circular chromosomes
Enzymes and structures involved in viral replication:
- viral replicase
- GP120
- GP41
- viral reverse transcriptase
- viral integrase
- viral protease
- host DNA polymerase
- host RNA polymerase
- host ribosomes
What is viral replicase?
a RNA dependent RNA synthesis
What is GP120?
glycoprotein involved in binding to the CD4 receptor during HIV
What is GP41?
glycoprotein involved in insertion into membrane and mediate fusion with host cell membrane
How does viral reverse transcriptase work during a viral infection?
- RNA dependent DNA synthesis makes a DNA copy of RNA viral genome
- DNA dependent DNA synthesis makes a DNA copy of DNA copied viral genome
- RNase activity will degrade RNA once DNA is copied
How does viral integrase work during a viral infection?
- cuts into host genome
- inserts DNA copy of viral genome
- seals into host genome
What happens with viral proteases during a viral infection?
- final processing of viral proteins once host cell begins producing enveloped viruses
- involved in the maturation of virus
What happens with host DNA polymerase during a viral infection?
copies host genome with newly integrated viral genome
What happens with host RNA polymerase during a viral infection?
transcribes host gene along with newly integrated viral genes
What happens with host ribosomes during a viral infection?
translates host proteins and viral proteins
6 classes of antiretroviral agents:
- nucleoside reverse transcriptase inhibitors (NRTIs)
- non-nucleoside reverse transcriptase inhibitors (NNRTIs)
- protease inhibitors (PIs)
- integrase inhibitors (INSTIs)
- fusion inhibitors (FIs)
- chemokine receptor antagonists (CCR5)
Function of NRTIs:
block reverse transcriptase activity by competing with active site for the substrate
Function of NNRTIs:
interfere with substrate binding to reverse transcriptase by distorting active site
Function of PIs:
interfere with maturation of virus
Function of INSTIs:
interfere with cutting of host sequence and insertion of the DNA copy of viral genome into host genome
Function of FIs:
block insertion of glycoprotein into the membrane
Function of CCR5 antagonists:
block binding of virus to host cell receptors
For transcription, what happens if there is a mutation?
most likely will change the efficiency of the promoter
What happens during initiation of transcription?
rate determining step
- sigma subunit recognizes promoter sequence at TATA box in promoter
- RNA polymerase binds to sigma and forms closed complex
- RNA polymerase forms open complex (transcription bubble)
What happens during elongation of transcription?
- short sequence of nucleotides are created (7-10)
- sigma subunit dissociates to increase stability of transcription complex
- elongation continues
Types of transcription termination:
- factor dependent: needs protein to knock RNA polymerase off (rho protein)
- factor independent: needs formation of an RNA hairpin in an area that blocks RNA polymerase
What happens during transcript processing, which occurs after transcription but before translation?
- addition of poly A tail and cap to start of sequence
- splicing
What happens during splicing?
- spliceosome mediates it
- introns removed (non-coding) and loops to form a lariat structure before exons are linked
- exons joined (coding)
The lac operon regulates transcription by…
turning on transcription when repressor protein is prevented from binding to operon
- presence of inducer = repressor can’t bind
The Trp operon regulates transcription by…
allows cell to express Trp synthesis enzyme by turning on when Trp levels are low
What happens to the Trp operon when Trp levels are high?
- enough Trp charged tRNA so ribosomes don’t pause when reaching the first Trp codon
- doesn’t allow 2-3 hairpin to form
- 3-4 hairpin forms and acts as terminator
What happens to the Trp operon when Trp levels are low?
- ribosomes reach Trp codons and waits for Trp charged tRNA
- allows formation of 2-3 hairpin (anti-terminator)
- Trp synthesis enzymes created
Steps of translation:
- 30S subunit binds the mRNA and alignment is ensured by initiation factors (IF) and Shine-Dalgarno sequence
- initiator tRNA binds to P site
- 30S binds with 50S subunit
Shine-Dalgarno sequence is utilized by what structure?
ribosomes
