Term
| What is the driving force of DNA replication? |
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Definition
| Hydrolysis of pyrophosphate created by beta and gamma phosphates. |
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Term
| How are nucleoside analogs used as drugs to inhibit DNA replication? |
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Definition
| They lack the 3'-OH group, acting as chain terminators. |
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Term
| Where does DNA replication begin in prokaryotes and eukaryotes? |
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Definition
Origin site - specific DNA sequence (AT rich) that binds specific proteins
Prokaryotes - single site origin (OriC)
Eukaryotes - multiple sites of origin |
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Term
| What directs the initiation process? |
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Definition
| Protein-DNA and protein-protein interactions. |
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Term
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Definition
| Unidirectional, energy-dependent enzyme that catalyzes unwinding, which creates positive supercoiling downstream. |
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Term
| What are single stranded DNA binding proteins and why are they important? |
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Definition
| Proteins that bind single stranded DNA and shift the equilibrium toward single stranded DNA and prevent attacks from nucleases; ssDNA is unstable and to prevent re-annealing of strands. |
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Term
| What is topoisomerase type 1 and type 2? |
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Definition
Type 1: Relaxes one supercoiled strand at a time by making a nick in one strand; ATP independent
Type 2: Relaxes both supercoiled strands by making a nick in each strand; ATP dependent. |
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Term
| How are topoisomerases used as drug targets? |
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Definition
Anti-cancer: stops replication by generating strand breaks in DNA
Anti-bacterial: inhibits gyrase |
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Term
| How is the lagging strand synthesized? |
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Definition
| 3' to 5' in Okazaki fragments of ~1000 nucleotides in prokaryotes and ~200 nucleotides in eukaryotes. |
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Term
| What does DNA polymerase do? |
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Definition
| Catalyzes the addition of nucleotides to the 3'-OH group of a growing strand. It cannot initiate the synthesis of a new strand. |
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Term
| What is the RNA primer and how often is it synthesized? |
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Definition
| A short stretch (~10 nucleotides) of RNA that provide the 3'-OH group for nucleotide addition; It is synthesized only once for the leading strand and it is constantly formed for the lagging strand. |
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Term
| What initiates replication? |
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Definition
Primase - a unique RNA polymerase
In eukaryotes, Pol alpha/primase complex |
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Term
| What are the 3 enzyme activities of DNA polymerase? |
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Definition
1. DNA synthetic activity - requires primer, template, Mg2+/Zn2+, and all dNTPs.
2. 3' to 5' exonuclease activity - removes mispaired bps - proofreading activity
3. 5' to 3' exonuclease activity - degrades RNA or DNA strands that are hybridized to template strand. (In prokaryotes this removes RNA primers) |
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Term
| What is the beta subunit of DNA polymerase? |
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Definition
| Beta subunit encircles the dsDNA, forming a sliding clamp. Responsible for the processivity of the polymerase enzyme. |
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Term
| How many polymerases in prokaryotes? Describe Pol I and Pol III. |
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Definition
5
Pol I - slow enzyme (~50 nucleotides/min) with all 3 enzyme activities. Mostly involved in RNA primer removal and DNA repair.
Pol III - High prossessivity (~9000 nucleotides/min); no 5' to 3' exonuclease; replicates most of DNA in E. coli |
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Term
| What is the role of DNA ligase? |
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Definition
| Seals ssDNA nicks; ATP dependent |
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Term
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Definition
| In prokaryotes, two circular DNA molecules linked in a chain are untangled by topoisomerase II. |
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Term
| Compare the intricacies of DNA replication in pro and eukaryotes. |
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Definition
| Eukaryotic replication is similar to prokaryotic, but it is slower and more complicated with more proteins. |
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Term
| How many eukaryotic DNA polymerases? Describe Pol beta, delta, and epsilon. |
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Definition
5 (alpha - epsilon)
Pol beta - similar to Pol I, functions in repair
delta - polymerase of lagging strand
epsilon - polymerase of leading strand |
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Term
| What is RNaseH responsible for? |
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Definition
| Removal of RNA primers in eukaryotes. |
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Term
| Describe polymerase switching in eukaryotes. |
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Definition
| Replication is initiated by Pol alpha/primase complex, which then incorporates a few nucleotides before being replaced by Pol delta or Pol epsilon. |
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Term
What is a telomere?
What is telomerase? |
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Definition
A long segment of repeated sequence creating the 3' overhang in DNA synthesis;
A reverse transcriptase (RNA dependent DNA polymerase) that adds the repeating units. |
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Term
Describe telomerase activity in somatic cells and immortal cells.
Describe telomerase activity in cancer. |
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Definition
somatic: lack telomerase activity, die after 20-60 divisions
immortal: have telomerase activity
cancer: over expressed in 90% of cancers |
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Term
What are the 2 types of mutations?
What is a mutagen?
What is a mutant? |
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Definition
Point mutation - change in single bp; Insertion or deletion - of single or multiple bps
Mutagen - mutation causing agent
Mutant - organism containing a mutation |
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Term
| What does polymorphism refer to? |
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Definition
| An alteration in DNA sequence that was not repaired. |
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Term
| What are the 3 endogenous sources of DNA damage? |
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Definition
1. Mismatches
2. Spontaneous alterations in chemistry such as C deaminating to U
3. Insertion by mobile elements such as transponsons. |
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Term
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Definition
| Repair of replication errors and small insertion or deletion. Mut proteins recognize the mismatch and make a nick in the newly synthesized strand. Mutation in mut protein homologs in humans leads to Hereditary Non-Polyposis Colon cancer and other spontaneous CA. In E. coli, methylation of parent strand is necessary for differentiation. |
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Term
| Hereditary Non-Polyposis Colon CA (HNPPCC) |
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Definition
| caused by defect in mut protein homologs - mismatch repair pathways |
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Term
| Base Excision Repair (BER) |
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Definition
| Repair of damaged bases or ssDNA breaks. Removal of damaged base by glycosylase, cleavage of the suger phosphate backbone by apyrimidine/apurine endonuclease, removal of abasic nucleotide by exonuclease, insertion of correct nucleotide by polymerase, and ligation of backbone. |
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Term
| Nucleotide excision repair (NER) |
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Definition
| Repairs a variety of helix-distorting DNA lesions such as pyrimidine dimer, bulky adduct on base and cisplatin-induced DNA damage. UVR proteins (E. coli) or XP proteins (humans) remove a short ss segment containing the defect. Gap is filled by DNA polymerase. |
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Term
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Definition
1-4 per million people; autosomal recessive; mutations in 7 XP genes, causing a defect in early NER
excessive sunburn, dry skin, excessive freckling, eye damage, keratosis; progressive neuro damage; developmental defects; 2000 fold higher rate of fatal skin CA
Avoid UV light |
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Term
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Definition
1 per million people; autosomal recessive; mutations in 2 genes of NER pathway; RNA synthesis following UV exposure is reduced
dwarfism and developmental delay; UV sensitive; retinal atrophy; deafness; Mickey Mouse ears; bird like pointed nose; deficient in subQ fat and premature aging; no increased risk for skin CA |
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