BIOL 2311 Chapter 14 (DNA Structure and Replication)

James Watson and Francis Crick discovery

model of DNA structure

Frederick Griffith, 1928

discovered transformation, experiments of Streptococcus pneumonia

transformation

alteration of a cell’s hereditary type by the uptake of DNA released by the breakdown of another cell

Griffith’s experiment

smooth (S) strain is virulent with capsule, rough (R) strain is nonvirulent with no capsule, mice died when injected with live R and heat killed S

determined that some factor (transforming principle) derived from dead S cells caused R cells to be converted to S cells

Oswald Avery, 1940s

identified chemical nature of Griffith’s transforming principle

broke down heat killed S bacteria and destroyed either protein, DNA or RNA

destroyed protein or RNA had no effect on R transforming into S

destroyed DNA stopped transformation from occurring - transforming principle is DNA

Alfred Hershey and Martha Chase, 1952

proved DNA is the hereditary molecule, studied a virus, “T2 phage,” that infects E. coli, virus is made of DNA/RNA surrounded by protein

protein shell labeled with radioactive 35S, DNA labeled with 32P

progeny phages had significant amounts of 32P and little of 35S

Lytic Cycle of Bacteriophage

attachment, penetration, biosynthesis, maturation, lysis

Lysogenic Cycle of Bacteriophage

after penetration, phage DNA is incorporated into host DNA, cell continues to mitosis, when stressed viral DNA is separated from host DNA, rest of lytic steps continue

Chargaff’s Rules

nitrogenous bases of DNA occur in definite ratios

# of purines = # of pyrimidines (adenine = thymine, guanine = cytosine)

X-ray diffraction

X ray beam is directed at a solid molecule, diffraction patterns are used to deduce atom positions, used by Maurice Wilkins and Rosalind Franklin to study DNA structure

Semiconservative replication

actual form of DNA replication, parental strands are separated and each are used to construct a new daughter strand - each daughter has one parental strand and one new strand

Conservative replication

whole parental molecule is one of the daughter molecule

Dispersive replication

small random pieces of parent molecule are in daughter molecules

Messelsohn and Stahl, 1958

demonstrated that DNA replication is semiconservative, tagged parental strands with “heavy” 15N, final density gradient proved semiconservative replication

1. E.coli cells grown in 15N medium, sample collected and DNA purified

2. cells transferred to 14N medium, after one cell division sample collected and DNA purified

3. cells divide one more time in 14N medium, sample collected and DNA purified

4. centrifuge to show density gradient

DNA polymerase structure

“hand shaped,” template DNA lies over palm, template strand and 3’-OH of new strand meet at active site in palm domain

when incoming nucleotide is added, thumb and fingers close over the site to facilitate the reaction

sliding DNA clamp

protein, encircles DNA, attaches to rear end of DNA polymerase, tethers DNA polymerase to template strand, increases rate of DNA synthesis

DNA helicase

unwinds DNA strands

Single stranded binding proteins (SSBs)

coat exposed single stranded DNA, stops them from pairing

topoisomerase

avoids twisting of DNA ahead of replication fork; cuts DNA turns DNA to opposite of twisting and rejoins (in circular DNA)

primase

creates a short chain of RNA (primer) so that DNA polymerase can start working

leading strand

new DNA strand is synthesized in the direction of DNA unwinding, DNA polymerase does not need to stop at any point

lagging strand

new DNA strand is synthesized discontinuously in the direction opposite of DNA unwinding, DNA polymerase stops and restarts multiple times, primes keeps making new primers

Okazaki fragments

the short segments of DNA that are created on the lagging strand

DNA ligase

binds Okazaki fragments together

DNA polymerase III

the main polymerase that synthesizes DNA

DNA polymerase I

replaces RNA primers with DNA in lagging strand

Direction of DNA synthesis

DNA is created in the 5’→3’ direction, new nucleotides added to 3’ end

therefore template strand is “read” in 3’→5’ direction

replisome

the whole protein complex responsible for DNA synthesis

origin of replication (Ori)

quite literally where DNA replication starts, 1 in bacteria, many in eukaryotic cells

telomeres

the ends of DNA strands, creates a buffer of noncoding DNA, telomere repeat

telomere repeat

TTAGGG

shortening of DNA

the original primer on the 5’ end cannot be replaced by DNA polymerase so chromosomes shorten with each replication

telomerase

stops shortening of telomeres by adding telomere repeats to chromosome ends, only active in rapidly dividing embryonic cells, germ cells, and cancerous somatic cells

1. RNA primer is removed

2. telomerase binds to 3’ end of template strand by complementary pairing in telomerase RNA

3. new telomere DNA is synthesized using telomerase RNA as the template

proofreading mechanism

during DNA synthesis

1. DNA polymerase recognizes mismatched base pair

2. enzyme reverses, uses 3’→5’ exonuclease to remove mispaired nucleotide

3. resumes polymerization in 5’→3’ direction

mismatch repair

1. mismatch repair proteins scan DNA for a mispaired base and cleaves region of new strand around the mismatch

2. repair DNA polymerase fills in the gap (5’→3’)

3. DNA ligase seals the nick

base-excision repair

non-bulky damage is repaired by removing the erroneous base and replacing it with the correct one

nucleotide-excision repair

bulky distortions (e.g. thymine dimer) are repaired by removing an entire segment of DNA

thymine dimer

adjacent thymines are fused together, distorting the DNA - caused by UV light radiation

primary source of mutations

errors left in DNA after proofreading and DNA repair, mutations are ultimate source of variability