DNA Replication

Owly Says:

This lesson is probably the most “memory” heavy in this unit so don’t be afraid to ask questions. (think of it as the “glycolysis lesson” of this unit)

History• How does DNA replicate?

– Does it split down the middle?• If so, do those strands come back together?• Are new strands formed there?

Meselsohn-Stahl Experiment

THE PROCESS

– relies on two basic steps, each mediated by cellular proteins and enzymes:

• UNWINDING & UNZIPPING

• the DNA molecule opens up to expose its bases– can happen from molecule’s end or anywhere along the length

(REPLICATION BUBBLES)– Rep. Bubbles help speed up DNA replication process

• NEW BASE PAIRING

– once old (conservative) bases are exposed, new nucleotides will automatically pair with them

– the old strands serve as a template for replication (a model, with “built – in” instructions)

– the new nucleotides are found free-floating in nucleoplasm

POTENTIAL PROBLEMS

• Untwisting and unzipping without breaking the DNA:

– HELICASE untwists the double helix (breaks H-bonds)

– GYRASE works with helicase to prevent breakage; it “swivels” the DNA slightly (by cutting and re-sealing the DNA backbone) to relieve tension before Helicase does its job

Keeping strands separated during new pairing

– SINGLE-STRANDED BINDING PROTEINS (SSB’s) bind to each exposed strand; sort of like a doorstop. (without them, the DNA would form a helix again, since base pairing is so automatic)

Forming the new strand

• PRIMASE prepares a primer of RNA as a foundation– DNA POLYMERASE III links new nucleotides

together to form phosphodiester bonds– DNA POLYMERASE I will eventually

destroy/replace primers

Dealing with directionality of DNA

– DNA must be synthesized in a 5’ to 3’ direction– This means each new strand is being made in opposite

directions– For the 3’-5’ template strand, its new partner is made in one

continuous piece; this is the LEADING STRAND– For the 5’-3’ template strand, its new partner is made in a stop &

go way: as the replication for keeps opening, more replication can occur, little by little

– This constant “catching up” gives this strand the name, LAGGING STRAND

– The lagging strand is composed of lots of small pieces of DNA called Okazaki Fragments which need to be joined together

– The enzyme LIGASE links together these fragments

Damage and errors in Replication

– DNA POLYMERASE I and III “proofread” the new DNA

– If a pairing error is found, they will back up, cut out the error and replace it

– This is an exonuclease function of these enzymes

Damage and Repair

• Any damage to DNA would be lethal. Cells often spend much more energy repairing DNA than synthesizing it.

• Correcting damage due to enviromental effects – Example: UV light thymine dimers. Energy in UV links thymine

where it occurs side-by-side on one strand of DNA, screws up the ability of this bit of DNA to serve as template for replication or for correct reading of proteins.

• One good 4-hour day at beach 10 UV-induced errors in DNA of every skin cell – Your skin cells spend lots of energy patrolling DNA, detecting

such errors, cutting them out, and using the remaining good strand as a template for repair synthesis.

• Correcting errors during replication (proofreading) – When new DNA is synthesized, occasional

errors in base pairing occur with frequency ~ 1 in 10,000 base pairs

– If not corrected, could lead to mutations, loss of functions, loss of competitiveness, evolutionary weeding out.

– Proofreading carried out by DNA polymerases enzymes; if base mismatch spotted, cut out new bases (keep track of which is template strand and which is new strand during replication), resynthesize copy strand from that neighborhood of template.