B3 01 DNA Replication Intro

DNA Synthesis Extends RNA Primers.

- Careful analysis of Okazaki fragments revealed that 5′ ends consist of RNA segments of 1 to 60 nt that are complementary to the template DNA chain. (Prime reaction and removed afterward) →In E. coli, these RNA primers are synthesized by the enzyme primase. - Multiple primers are required for lagging strand synthesis (discontinuous), but only one primer is required to initiate synthesis of the leading strand. - Mature DNA does not contain RNA. - The RNA primers are replaced with DNA!!! :)

DNA Replication

- DNA is replicated by enzymes DNA-directed DNA polymerases - Use single-stranded DNA (ssDNA) as templates - Synthesis of the complementary strand (Watson-Crick base pairs) from the appropriate deoxynucleoside triphosphates (dNTP) - Nucleophilic attack of the growing DNA chain's 3'-OH group* - Reaction is driven by the subsequent hydrolysis of the eliminated PPi. - DNA chains are extended only in the 5' → 3' direction (reading 3' to 5')

DNA Polymerase Senses Watson-Crick Base Pairs via Sequence-Independent Interaction

- DNA polymerase must distinguish correctly paired bases from mismatches and do so by sequence-independent interactions with the incoming dNTP - Recognition occurs in an active site pocket - bound dsDNA is mainly in the B conformation, the 3 base pairs nearest the active site assume the A conformation→The resulting wider and shallower minor groove (A DNA) permits protein side chains to form hydrogen bonds in a sequence-independent manner - The polymerase also makes extensive sequence-independent hydrogen bonding and van der Waals interactions with the DNA's sugar-phosphate backbone. -Open conformation of pol binds to one base then transitions to a closed conformation upon binding the correct dNTP

Replication Is Semidiscontinuous

- Duplex DNA's two antiparallel strands are simultaneously replicated at a replication fork. - The two parent strands are replicated in different ways! - Leading strand is continuously synthesized in its 5' → 3' direction as the replication fork advances. - The lagging strand is also synthesized in its 5' → 3' direction →However, it can only be made discontinuously, as Okazaki fragments, as single-stranded parental DNA becomes newly exposed at the replication fork. - The Okazaki fragments are covalently joined together by the enzyme DNA ligase.

DNA Polymerases Add the Correctly Paired Nucleotides (Processive enzyme)

- E. coli (PROKARYOTE) enzyme that catalyzes the synthesis of DNA is now known as DNA polymerase I or Pol I, consists of a single 928-residue polypeptide. - A Processive enzyme because it catalyzes a series of successive nucleotide polymerization steps,20 or more, without releasing the single-stranded template. [- Although Pol I was the first of the E. coli DNA polymerases to be discovered, it is not E. coli's primary replicase.]

Pol I 3' → 5' exonuclease

- In addition to polymerase activity, Pol I has *two independent hydrolytic activities that occupy separate active sites: a 3' → 5' exonuclease and a *5' → 3' exonuclease. - The 3' → 5' exonuclease activity→allows Pol I to edit its mistakes. (blue-correcting, go backwards and scans) [we are polymerizing 5' to 3'] - If Pol I erroneously incorporates a mispaired nucleotide at the end of a growing DNA chain, polymerase activity is inhibited and the 3' → 5' exonuclease hydrolytically excises the nucleotide. - Polymerase activity resumes DNA replication. This *proofreading mechanism explains the high fidelity of DNA replication by Pol I.

Pol I Has Exonuclease Activity

- Remove the RNA primers and *replaces them with DNA* - Its most important (and only essential) function is in lagging strand synthesis - Involves the 5' → 3' exonuclease activity at the 5' end of the single-strand nick between the new and old (previously synthesized) Okazaki fragments - Replace them with deoxynucleotides that are appended to the 3' end of the new Okazaki fragment. - The nick is thereby translated (moved) toward the DNA strand's 3' end, a process known as nick translation. - Nick is sealed by the action of DNA ligase, linking the new and old Okazaki fragments.

Pol I 5' → 3' exonuclease

- The Pol I 5' → 3' exonuclease binds to duplex DNA at single-strand nicks (breaks)→It cleaves the nicked DNA strand in a base-paired region *beyond the nick* to excise the DNA as either mononucleotides or oligonucleotides of up to 10 residues.

DNA Replication Occurs at Replication Forks.

- circular chromosomes contain replication "eyes" or "bubbles" called θ structures - Replication fork = branch point; DNA synthesis - θ replication is almost always bidirectional.

The DNA Polymerase Catalytic Mechanism Involves Two Metal Ions

Active sites all contain two metal ions, usually Mg2+, that are liganded by two Asp side chains Metal ion B: -liganded by all three phosphate groups of the bound dNTP -orients its bound triphosphate group -electrostatically shields its negative charges Metal ion A -bridges the α-phosphate group of this dNTP and the primer's 3'-OH group -activates the primer's 3'-OH group for a nucleophilic attack on the α-phosphate group

Pol I also functions in the repair of damaged DNA

Pol I's 5' → 3' exonuclease activity then excises the damaged DNA -Same version of nick translation