Exam 4- Enzymes that process the genome; DNA replication is semi-conservative; DNA polymerases

what are the 3 enzymes involved in polynucleotide synthesis?

- DNA polymerase
- RNA polymerase
- reverse transcriptase

DNA polymerase

- adds one deoxyribonucleoside monophosphate (dNMP) to 3' end of a DNA chain
- requires a DNA template chain and a primer
- primer must have a 3'-OH

RNA polymerase

- adds one ribonucleoside monophosphate (NMP) to 3' end of an RNA chain
- requires a DNA template chain
- can initiate de novo synthesis of RNA

reverse transriptase

- adds one dNMP to 3' end of DNA chain
- requires an RNA template chain and a primer

what is able to digest polynucleotide chains?

nucleases

______ is specific for DNA while ____ is specific for RNA

DNase; RNase

exonuclease

- breaks a phosphodiester bond at ONE END of a polynucleotide chain
- either 5' to 3' or 3 to 5'

endonuclease

- breaks phosphodiester bond WITHIN a polynucleotide chain
- sequence independent/specific
- single strand/ double strand break

excinuclease

breaks 2 phosphodiester bonds within a SINGLE polynucleotide chain

what are restriction endonucleases/enzymes?

- type of endonuclease that only breaks phosphodiester bonds at a specific DNA sequence (restriction sites)

what are palindromic sequences?

Sequences that consist of base pairs that read the same in the opposite direction

true or false: most restrictive enzymes recognize palindromic sequences

true

what is the only enzyme that can link two existing DNA chains by forming a phosphodiester linkage?

DNA ligase

recombinant DNA technology

DNA processing new enzymes that can be used to create new combinations of sequences

what does in vitro DNA ligase do?

- joins two DNA molecules together end-to-end
- joins blunt-ends or compatible sticky-ends created by endonucleases

conservative replication

yields an original intact DNA molecule and one entirely newly synthesized DNA

semi-conservative replication

yields two DNA molecules, each with one parental and one newly synthesized strand

dispersive replication

yields two DNA molecules that are hybrids (or mixtures) of parental and newly synthetized DNA

explain the meselson and stahl experiment

1. E.coli grown in heavy N15; a band forms at the bottom of the tube
2. switches to N14 medium, allowing one round of replication and division
3. only one band in the middle of the tube= part old DNA and part new= NOT conservative
4. repeated step 2; one band in the middle and one at the top of the tube= semi-conservative model true

what does a DNA polymerase with roman numerals represent?

they are from bacteria

what does a DNA polymerase with greek letters represent?

they are from eukaryotes

DNA polymerase _____ DNA synthesis

catalyzes

where is the catalytic site for DNA synthesis located?

the palm of the enzyme

Describe the mechanism of DNA polymerase

1. DNA polymerase, the enzyme, catalyzes the extension of a DNA strand one dNMP at a time
2. DNA synthesis starts; The Mg2+ ions facilitates attack of the 3'-hydroxyl group of the primer on the α phosphate of the nucleotide triphosphate
3. the other Mg2+ ion facilitates displacement of the pyrophosphate
4. bond breaks and releases B and y as a pyrophosphate

what makes sure that Mg2+ is where it needs to be at the catalytic site?

aspartate

an error in replication can introduce a _____ into the genome. It will be permanent and inherited by subsequent _____ cells

mutation; daughter

what is the average E. coli mutation rate?

- 1 bp in 10^9-10^10 bp
- an error occurs only per 1,000-10,000 replications

how are errors in replication fixed in all polymerases?

- DNA polymerase active site restricts base paring to watson-crick-franklin bp
- "geometry"; if base pairs do not fit into the active site, there is presynthetic error control and they start over

True or false: all polymerases go through proofreading

false

what are the 2 active sites for high-fidelity DNA polymerases?

- a catalytic site for DNA synthesis
- a 3' to 5' exonuclease site for removing mis-incorporated nucleotides

process of proofreading (think of a keyboard and backspace)

1. polymerase mispairs
2. repositions mispaired 3' terminus into 3' to 5' exonuclease site
3. exonuclease hydrolyses mispaired group
4. 3' terminus repositions back to polymerase site
5. polymerase incorporates the correct nucleotide

what are the 2 main prokaryotic DNA polymerases?

DNA polymerase I, DNA polymerase III

what are the cellular roles of DNA polymerase I and III?

genomic replication and DNA repair

compare and contrast DNA polymerase I and III in terms of proofreading, 5'-3' exonuclease, polymerization rate and processivity

- proofreading: both can
- 5'-3' exonuclease: I can, III can't
- polymerization rate: I is lower, III is higher
- processivity: I is lower, III is higher