DNA Replication

List three properties of DNA replication

replication is semiconservative, replication begins at origin and proceeds bidirectionally, and synthesis of new DNA occurs in 5’→3’ direction

In what direction does the synthesis of new DNA occur?

5 → 3

experiment that showed that nitrogen used for synthesis of new dsDNA becomes equally divided between the two daughter genome

• suggests semiconservative replication mechanism → each new DNA has one old (parent) and one new (daughter strand)

Meselson-Stahl

experiment showed circular PNA w/ extra loop, that both strands are replicate simultaneously, with two replication forks = bidirectional replication

Cairn’s

experiment showed that DNA replication loop always initiate at unique origin

• mapped the bubbles (denatured DNA at A=T rich regions)

Inman’s

leading and lagging strand are in ____ direction

5’ → 3’

nucleases that cleaves bonds that remove nucleotides from ends of DNA

exonucleases

nucleases that cleaves bonds within DNA sequence

endonucleases

is enzyme responsible for DNA synthesis

DNA Polymerase

short strand complementary to template that contains a 3’-OH to begin the first DNA polymerase -catalyzed reaction

• can be made of DNA or RNA (more common)

primer

Sequence of DNA polymerization

initiation → primer → elongation → processivity → proofreading → termination

• _______ site : where incoming nucleotide binds

• _______ site: newly made base pair resides when new polymerase moves forward

insertion, post-insertion

number of nucleotides added before dissociation

- each polymerase has own rate

processivity

_____"proofreads" synthesis for mismatched base pair

• translocation of enzyme to next position is inhibited until enzyme can remove

  the incorrect nucleotide

3' → 5' exonuclease

DNA polymerase ___

• abundant but not ideal for replication (slow rate, low processivity)

• primary function is clean up

I

DNA polymerase ___

• principal replication polymerase

III

DNA polymerase ___

• DNA repair

II, IV, V

DNA polymerase I also has _____ exonuclease activity

• DNA polymerase I moves ahead of 5’ → 3' exonuclease to hydrolyze & do

  nick translation (strand break move along with enzyme)

• activity is in the Klenow fragment (distinct domain that can be separated by

  protease treatment

5' →3'

List enzymes in replisome

helicases, topoisomerases, primases, DNA ligases

enzyme in replisome that uses ATP to unwind DNA strands

helicases

enzyme in replisome that relieves stress caused by unwinding

topoisomerases

enzyme in replisome that makes RNA primer

primases

enzyme in replisome that seals nicks between sucessive nucleotides on same strands (Okazaki fragments)

DNA ligases

PNA-A are _____ - initiator proteins

• 8 DNA-As bind to R and I sites → DNA wrap around complex (form supercoil)

• ↳ strain leads to denaturation of nearby due sites + associated proteins facilitate DNA bending

ATPases

relieves topological stress ahead of replication forks

DNA gyrase

hemimethylate OriC after replication ; fully methylate DNA to allow new Dna-A to bind

• hemimethylated OriC interacts w/ plasma membrane using SeqA

• SeqA dissociates & OriC Sequence is released from membrane

DAM methylase

in the process of strand elongation, ______ makes RNA primer

primase DNA-G

in the process of strand elongation, _____ addes nucleotide. to 3’ end of strand

DNA polymerase III

In the process of transitioning between Okazaki fragments, ____ removes RNA primer; fills in gap

DNA polymerase I

In the process of transitioning between Okazaki fragments, ____ seals the backbone between Okazaki fragment

DNA ligase

(is Type II Isomerases): separates interlinked DNA molecules

DNA topoisomerase IV