DNA & Genomics (DNA replication)

scrutinise nucleotide against template as soon as it is added to the growing strand -> DNA polym. removes the incorrectly paired nucleotide & resume synthesis (proofreading)

evidence for semi-conservative replication

1) E.coli grown in a medium containing NH4Cl with heavy nitrogen isotope N15 for many generations

2) Bacteria transferred into a medium containing lighter isotope of nitrogen, N14 & allowed to grow

each time cell divides, its DNA replicates & N15 is incorporated into nucleotides which are used to synthesise new DNA

at various times of 1, 2 or more generations, samples of bacteria were collected

3) DNA from each grp of bacteria was extracted & put into a caesium chloride solution & spun at 40000g in a centrifuge

4) caesium chloride molecules sink to the bottom, creating a density gradient.

DNA molecules position at their corresponding level of density whr its density equals to caesium chloride solution

5) Centrifuge tubes were observed under UV rays -> DNA appeared as fine bands in the tubes at diff. heights according to their density

DNA molecules containing N15 are heavier -> end up nearer the base of the tubes

results

6) By semi-c replication, DNA of 1st generation would be of intermediate density

all DNA molecules comprise 1 N15 strand & 1 N14 strand

7) Half of DNA molecules from 2nd generation would be of intermediate density & half would be of light density

50% of DNA molecules comprise 1 N15 strand & 1 N14 strand and 50% comprise 2 N14 strands

STAGE 1 : Unwinding

a portion of double helix unwind & unzip at the origin of replication by DNA helicase using energy from ATP

2 parental strands are separated by breaking hydrogen bonds btw nitrogenous bases

replication begins at origins of replication

a replication bubble is formed with 2 replication forks

DNA replication proceeds in both directions from each origin of replication

prokaryotes : only a single OoR

eukaryotes : multiple OoR

each strand is bound & stabilised by single-stranded binding proteins -> prevent them from rewinding behind the replication fork

unwinding of double helix causes tighter twisting & strain in front of replication fork -> positive supercoil

DNA topoisomerase introduces a break in a single strand -> allows the strand to rotate around the break & reseals the strand

eliminates the positive supercoil

each parental strand acts as a template for the synthesis of daughter strand

STAGE 2 : Priming

RNA primers are short segments of RNA (about 5-10 nucleotides) reuiqred for DNA polymerase to initiate elongation as RNA primers provide 3' OH end

primase (specialised RNA polym.) binds to the ssDNA template & synthesises RNA primers in the 5' to 3' direction

ribonucleotides are added one at a time via complementary base pairing using the parental DNA strand as a template on both sides of replication fork

ssBP are displaced whr the RNA primers are

STAGE 3 : Elongation

before start of DNA replication, free deoxyribonucleotides are synthesised in cytoplasm & transported into nucleoplasm via nuclear pores

DNA polym. adds deoxyribon. to the free 3' OH end of RNA primer as its active site is specific for -OH grp on the nucleotide

DNA polym. catalyses the synthesis of new strand of DNA in 5' to 3' direction via complementary base pairing with the parental strand

DNA polym. catalyses the formation of a phosphodiester bond btw the 3' OH end of primer & 5' phosphate grp of the dNTP added

leading strand

daughter strand synthesised continuously towards replication fork

lagging strand

daughter strand synthesised discontinuously via a series of Okazaki fragments away from replication fork

only 1 primer is required

each Okazaki fragment is primed separately

RNA primers are excised & replaced with deoxyn. by another DNA polym.

DNA ligase catalyses the formation of phosphodiester bonds btw 3' OH end of one fragment & 5' phosphate end of the previous fragement

STAGE 4 : Termination

product of replication is 2 daughter DNA molecules formed from 1 parental DNA molecule

each daughter DNA molecule is identical to the original parental molecule

each daughter molecule contains 1 strand conserved from the parental molecule & 1 newly synthesised strand

end-replication problem

DNA polym. complex is unable to completely replicate to the end of the chromosome

DNA polym. requires a free 3'OH grp to add deoxyribon. to

RNA primers at 5' end of newly synthesised strands are excised but cannot be replaced due to absence of 3' OH for polymerisation reaction

newly synthesised strand has a shorter 5' end due to the removed primers, while the parental template strand at 3' end is longer (single-stranded 3' overhang)

repeated rounds of DNA replication produce shorter & shorter DNA molecules

importance of base-pairing & hydrogen bonding in DNA

stability of DNA molecule

hydrogen bonds + hydrophobic interactions btw stacked bases stabilise the structure of double helix

adj. nucleotides within each polyn. strand are held tgt by strong covalent phosphodiester bonds -> not easily broken

DNA replication

double helical structure of DNA enables semi-c. replication

DNA polym. might improve specificity of complementary base pairing at 2 stages

scrutinise incoming nucleotide for proper complementarity with the template (pre-synthetic error control)

scrutinise nucleotide against template as soon as it is added to the growing strand -> DNA polym. removes the incorrectly paired nucleotide & resume synthesis (proofreading)

DNA repair

DNA is subjected to environmental factors that can cause changes in base sequence (mutations)

intact complementary strand can be used as a template to guide the repair by DNA repair enzymes

integrity of base sequence of DNA molecule is maintained