Lecture 3 - Discovery of DNA Polymerase and MS Flashcards
what was kornberg’s experiment?
he adds radioactive labels, denatures and precipitates DNA, then runs it thru a filter, and then measures radioactivity (radioactivity was in eluate)
what did kornberg find?
he found DNA Pol 1
How is DNA elongated by DNA Pol?
uses parental strand as template, uses nucleoside triphosphates as substrates
what is the mechanism of that DNA Pol facilitates?
nucleophilic OH group on 3’ attacks alpha-phosphate from the triphosphate, pyrophosphate is good leaving group since it stabilized by beta and gamma phosphates; need OH 3’ group and Mg2+ ions (help make OH more nucleophilic)
what were the 3 different models of DNA replication?
what was the meselson and stahl experiment?
cells were grown in N15 until all labeled, then grown on N14 and hybrid strands (one band), then grown in 14N again now there are hybrids and 14N strands (2 bands) = DNA replication is semiconservative
what was cairns experiment?
radiolabeled and it shows that DNA is replicated at the same time bidirectionally
what was inman’s experiment?
denaturation of DNA at A-T rich regions and happened at the same place
what activities does DNA Pol 1 have?
polymerase activity (extension of 5’ to 3’)
3’ to 5’ exonuclease activity (proofreading)
5’ to 3’ exonuclease activity (nick translation)
what are the different parts of DNA Pol 1?
2 fragments: large fragment contains klenow fragment which has polymerase activity and 3’ to 5’ exonuclease activity (proofreading); small fragment has 5’ to 3’ exonuclease activity (nick translation)
What is the mech of 3’ to 5’ exonuclease activity of klenow fragment?
cleavage happens at phosphodiester bond in a way that the 3’ OH can regenerate to continue DNA synthesis
how does proofreading work?
when polymerase activity makes a mistake, the large fragment stops. the delay allows for the 3’ end to move into the 3’ exonuclease site and excised
what is the process of 5’ to 3’ nick translation?
when enzyme finds a nick, it degrades DNA in a 5’ to 3’ and adds NT in a 5’ to 3’ moving the nick; good for removing primers and repairs
what are properties of DNA Pol 1?
enzyme is very slow, enzyme is not processive (falls off DNA shortly), present in high amounts which not consistent with origin of replication, not required for DNA synthesis
what is DNA replicase?
responsible for chromosome replication
comparison of 3 DNA Pol
