Chapter 2: DNA Replication and Repair Flashcards
Polymerases?
enzymes that synthesize nucleic acids by forming phosphodiester (PDE) bonds
Nucleases? (moreso MOA)
enzymes that hydrolyze PDE bonds
Exonucleases?
remove nucleotides from the 5’ or the 3’ end of a nucleic acid
Endonucleases?
cut within the nucleic acid and release nucleic acid fragments
Is the p arm of the chromosome the long or short arm?
short arm
Is the q arm of the chromosome the long or short arm?
long arm
High fidelity synthesis occurs with RNA polymerase or DNA polymerase?
DNA polymerase
Low fidelity synthesis occurs with DNA or RNA polymerase?
RNA polymerase
Do you need primer for RNA polymerase?
no primer needed
Compare and contrast require substrates for DNA or RNA polymerase?
dATP, dGTP, dCTP, dTTP (for DNA polymerase)
ATP, GTP, CTP, UTP (for RNA polymerase)
Do you require primer for RNA polymerase?
no
Is there proofreading activity with RNA polymerase? DNA polymerase?
no
yes
List the steps in DNA replication. (prokaryotes)
- Base sequence at the origin of replication is recognized
- Helicase breaks the H bonds holding the base pairs together. This allows the 2 parental strands of DNA to begin unwinding and forms 2 replication forks
- ssDNA binding protein (SSB) binds to the single stranded portion of each DNA strand, preventing them from reassociating and protecting them from degradation by nucleases
- Primase synthesizes a short (about 10 nucleoties) RNA primer in the 5’ -> 3’ dir., beginning at the origin on each parental strand; parental strand is used as template.
- DNA polymerase III begins synthesizing DNA in the 5’ -> 3’ direction, beginning at the 3’ end of each RNA primer. Strand can be made continuously in one long piece (“leading strand”)
- RNA primers are removed by RNAase H in eukaryotes and an uncharacterized DNA polymerase fills in the gap with DNA; in prokaryotes, DNA polymerase I both removes primer (5’ exonuclease) and synthesizes new DNA, beginning at the 3’ end of the neighboring Okazaki fragment
- Both eukaryotic and prokaryotic DNA polymerases proofread
- DNA ligase seals the “nicks” between Okazaki fragments, converting them to. a continuous strand of DNA
- DNA gyrase (DNA topoisomerase II) provides a “swivel” in front of each replication fork. As helicase unwinds DNA at replication fork the DNA ahead of it becomes overwound and positive supercoils form. (DNA gyrase inserts negative supercoils be nicking both strands of DNA, passing DNA strands through the nick, and then resealing both strands.)
Why are RNA primers required for DNA polymerase?
because DNA polymerases are unable to initiate synthesis of DNA can only extend a strand from the 3’ end of a preformed “primer”
What is the lagging strand?
synthesized discontinuously as series of small fragments known as Okazaki fragments
Each Okazaki fragment is initiated by synthesis of an RNA primer by a primase, and then completed by synthesis of DNA using what enzyme?
DNA polymerase III
Purpose of RNAase H?
5’ exoribonuclease acivity
digest the RNA primer from fragment 1
In eukaryotic cells, DNA polymerase extends the next fragment to fill in the gap
In prokaryotic cells DNA polymerase 1 has both the 5’ exocnulease acitivity to remove primers, and the DNA polymerase acitivty to extend the next fragment to fill in the gap
In both types of cells DNA ligase connects fragments 1 and 2 by making a phosphodiester bond
What is telomerase?
completes replication of the telomere sequence at both ends of eukaryotic chromosome
Where is telomerase present?(What type of cells)
embryonic cells, fetal cells, and certain adult stem cells;
inappropriately present in many cancer cells
Where is telomerase not present?
in adult somatic cells
What are telomeres?
repetitive sequences at ends of linear DNA moelcules in eukaryotic chromsomes
Relate telomeres and aging of cells?
with each round of replication in most normal cells the telomeres are shortened because DNA polymerase cannot complete synthesis of the 5’ end of each strand
Compare and contrast origin of replication for prokaryotes and eukaryotes.
Prokaryotes:
one ori site per chromsome
Eukaryotes:
multiple ori sites per chromsome
Compare and contrast how unwinding of DNA double helix is performed in prokaryotes and eukaryotes?
helicase in both
Compare and contrast stabilization of unwound template strands in prokaryotes and eukaryotes?
single-stranded DNA-binding protein (SSB) in both
Compare and contrast synthesis of RNA primers in prokaryotes and eukaryotes?
primase in both
Compare and contrast synthesis of DNA leading strand, lagging strand (Okazaki fragments) in prokaryotes and eukaryotes?
Prokaryotes:
DNA polymerase III
DNA polymerase III
Eukaryotes:
DNA polymerase a + delta
DNA polymerase a + delta
Compare and contrast removal of RNA primers in prokaryotes vs eukaryotes?
Prokaryotes:
DNA polymerase I
(5’ -> 3’) exonuclease)
Eukaryotes:
RNAase H
(5’ -> 3’ exonuclease)
Compare and contrast replacement of RNA with DNA in prokaryotes and eukaryotes?
DNA polymerase I (prokaryotes)
DNA polymerase δ (eukaryotes)
Compare and contrast joining of Okazaki fragments in prokaryotes and eukaryotes?
DNA ligase
Compare and contrast removal of positive supercoils ahead of advancing replication forks?
Prokaryotes
DNA topoisomerase II (DNA gyrase)
Eukaryotes
DNA topoisomerase II
Compare and contrast synthesis of telomeres in prokaryotes and eukaryotes.
Prokaryotes:
none
Eukaryotes:
telomerase
Some current uses of quinolones?
treatment of gonorrhea and upper and lower UTI in both sexes
What is reverse transcriptase?
RNA dependent DNA polymerase that requires an RNA template to direct the synthesis of new DNA
DNA synthesis by reverse transcriptase in retrovirusis can be inhibited by what?
AZT (zidovudine)
ddC (zalcitabine) 2’-3’ deoxycytidine
ddI (didanosine) 2’-3’ dideoxyinosine
AZT full name?
3’-azido-2’, 3’-dideoxythymidine
AZT MOA?
enters cell and can be converted to the triphosphate derivative and used as a substrate for the viral reverse transcriptase in synthesizing DNA from RNA genome
one replacement of an azide instead of normal hydroxyl group at the 3’ position of deoxyribose prevent further replication by effectively causing chain termination.
Does reverse transcriptase have proofreading activity?
Although it is a DNA polymerase, reverse transcriptase lacks proofreading activity
Cause of thymine dimers?
UV radiation
What is the recognition/excision enzyme in thymine dimers? In what condition is this enzyme deficient?
excision endonuclease (deficient in xeroderma pigmentosum)
Repair enzymes to fix thymine dimers?
DNA polymerase and DNA ligase
What part of the cell cycle can thymine dimers be created?
G1
What stage of the cell cycle can mismatched bases occur?
G2 and S
Cause of mismatched bases?
DNA replication errors
Examples of enzymes involved in pathogenesis of mismatched bases?
a mutation on one of two genes, hMSH2 or hMLH1, initiates defective repair of DNA mismatches, resulting ina condition known as hereditary nonpolyposis colorectal cancer- HNPCC
Repair enzymes for mismatched bases?
DNA polymerase and DNA ligase
Cytosine deamination occurs at what stage of the cell cycle?
G1
Cytosine deamination is caused by what?
spontaneous/ heat
What are the recognition/excision enzymes involved in cytosine deamination?
uracil glycosylase AP endonuclease
What are the repair enzymes in cytosine deamination?
DNA polymrase and DNA ligase
Name 3 tumor suppressor genes?
p53, ATM, Rb
Li Fraumeni syndrome and many solid tumors are associated with inactivation or deletion of what gene?
p53
MOA p53 gene?
encodes a protein that prevents a cell with damaged DNA from entering the S phase
ATM gene MOA?
encodes a kinase essential for p53 activity
ATM is inactivated in what condition?
ataxia telangiectasia, BRCA-1, and BRCA-2
Ataxia telangiectasia characteristics.
Hypersensitivity to X-rays and predisposition to lymphomas
Rb gene MOA?
negative regulator of the cell cycle through it ability to bind the transcription factor E2F and repress transcription of genes required for S phase.
What are microsatellites also known as?
short tandem repeats
What are microsatellites? How can they vary in size?
di, tri, or tetranucleotide repeats dispersed throughout the DNA, usu. (but not exclusively) in noncoding regions
for example, TGTGTGTG may occur at a particular locus. If cells lack mismatch repair, the replicated DNA will vary in the number of repeats at that locus e.g, TGTGTGTGTGTG or TGTGTG
Characteristics of XP?
extreme sensitivity to sunlight, skin freckling, ulcerations, and skin cancer… carcinomas and melanomas appear early in life
How can XP be diagnosed?
measurement of the relevant enzyme excision endonucleases in white cells of the blood