Overview of DNA replication Flashcards
What is the main enzyme in DNA replication
DNA polymerase is the main enzyme in DNA replication
Briefly explain the main function of DNA polymerase
DNA polymerase adds dNTPs(deoxynucleoside triphosphates) to an existing polynucleotide. It requires an oligonucleotide primer.
Does DNA replication occur simultaneously?
The two DNA strands are replicated simultaneously in the 5’-3’ direction.
Are primers RNA or DNA
Primers are RNA
Which enzyme(s) synthesize the primers
Both RNA polymerase and a special RNA polymerase called primase synthesizes primers in E.coli.
Briefly explain the life of a primer
In E.coli, the primer is synthesized by both RNA polymerase and primase. Then DNA polymerase extends the primer by adding dNTPs, eventually, the primer is excised and replaced by DNA.
Why does the cell have this extra complexity - the primer
The cell makes many copies of RNA, so it occasionally makes mistakes(mutations) but if a mutation occurs in DNA it could possibly be passed on to the cell’s progeny. A Watson-Crick base pair is partially stabilized by its neighboring base pairs (a cooperative interaction), the first few base pairs that are formed in a newly synthesized polynucleotide will initially be less stable than the base pairs that are formed later. Therefore, the first few base pairs are more likely to be erroneously incorporated due to mispairing than those at the end of a longer chain. If a primer were DNA then there would be no way to differentiate it from other DNA so that it can be replaced by more accurately synthesized DNA. But, the primer is RNA so it is readily identified and replaced.
Explain how both DNA strands are replicated simultaneously
DNA replication occurs simultaneously and this occurs at the replication fork. This is the junction where the two strands of the parental DNA are separated and the daughter strands are synthesized, each by different molecules of DNA polymerase. One of the DNA polymerases continuously copies the parental strand that extends in the 3’-5’ direction from the replication fork, thereby synthesizing the leading strand in the 5’-3’ direction. The other DNA polymerase also synthesizes in the 5’-3’ direction but it does so discontinuously- it binds the looped-around strand lagging strand template(5’-3’) so as to extend the RNA primer in its 5’-3’ direction until it encounters the previously synthesized primer. The DNA polymerase then disengages from the lagging strand template and rebinds upstream of the previous position, where it then extends the next primer to be synthesized.
What is the difference between the leading and lagging strand
The leading strand (5’-3’) is synthesized continuously and the lagging strand (3’-5’) is synthesized discontinuously.
Describe the enzymes involved in synthesizing the primers in E.coli
In E.coli, the synthesis of primers on the lagging strand are catalyzed by primase and on the leading strand, it is catalyzed by both RNA polymerase and primase.
Name and describe the two species of DNA polymerase that E.coli contains
DNA polymerase III synthesizes the leading strand and most of the lagging strand. DNA polymerase I, which removes primers and replaces them with DNA (has 5’-3’ exonuclease activity). The 5’-3’ exonuclease function binds to single-strand nicks then excises a 1-10 nucleotide segment of the nicked strand in the 5’-3’ direction past the nick. DNA polymerase’s 5’-3’ exonuclease activity and polymerase activity work in concert, so as the 5’-3’ exonuclease activity removes the primer its polymerase activity replaces the RNA with DNA.
Describe the completion of the leading and lagging strand synthesis.
The synthesis of the leading strand is complete with the replacement of its single RNA primer with DNA. The synthesis of the lagging strand requires the nicks between the multiple discontinuously synthesized fragments to be sealed. An enzyme called DNA ligase covalently links adjacent 3’-OH and 5’-phosphate groups.
What is the error rate of DNA polymerase and RNA polymerase in E.coli
RNA polymerase has an error rate of 1 in 10^4 nucleotides it transcribes and DNA polymerase has an error rate of 1 in 10^8-10^10 nucleotides.
What is the main reason for the enormous fidelity in DNA replication
Both Pol I and III have 3’-5’ exonuclease activity. The 3’-5’ exonuclease activity degrades newly synthesized 3’-end of a daughter strand one nucleotide at a time. This enzymatic function is activated by Watson-Crick base pairing and acts to edit out the occasional mistakes made by the polymerase function.
