DNA structure and replication (4) Flashcards
Why were proteins believed to be the genetic material?
diversity of their composition (20 amino acids) and their presence in chromosomes
Fredrik Griffith
used pneumonia causing bacteria on mice, concluded that a strain could transfer its genetic material to another strain
Oswald Avery, Colin McLeod, Maclyn McCarty
used DNase, RNase, and protase on strands in in vitro study. Found that the sample with DNase was the only one that did not transfer its genetic material to another
Bacteriophages
viruses infecting bacterial
Alfred Hershey and Martha Chase
used varuses and labeled dna and proteins. injected to bacterial.
centrifuged, found the pellet fraction contained most of the dna label.
concluded dna was the genetic material injected
Linus Pauling
proposed dna had a triple helix structure
dna
-nucleotide bases
-covalent bonds in a polymer
dna double helix 1
- dna consists of two strands that are h-boded together
dna double helix 2
- the two strands turn right to make a right-handed helix
dna double helix 3
the hydrophilic sugar phosphate is on the outside of the helix,
the negatively charged p groups make DNA souble in aqueous solutions
dna double helix 4
the hydrophobic nitrogenous bases are staked inside in a perpendicular manner to the strand. there are 10 bases per turn
dna double helix 5
-complimentary base pairing
- adenine pairs with thymine with 2 H-bonds and guanine pairs with cytosine with 3 H-bonds
- satisfices charagaff’s findings
dna double helix 6
- the two strands run in oppositite directions (anti-parallel orientation)
- The starting of dna has a phosphate group attached to the 5th C of ribose
- The other end has a -OH group attached to the 3rd C of ribose
dna double helix 7
the helical turns make a major groove and a minor groove between the adjacent turns
dna double helix 8
dna stores information in its sequence of bases
dna double helix 9
based on the complimentary base pairing, dna replication was proposed to be semiconservative
- the new copy is made from a template dna with high specificity
Where does supercoiled dna occur?
in circular dna (plasmids, bacterial chromosomes, chloroplast, and mitochondrial dna) and linear eukaryotic chromosomes
Why is supercoiling important?
Important to keep dna compact in the cell and during dna replication
Where is single-stranded dna present?
viral genomes
Why is the double strandedness of dna important?
important for stability and proofreading during replication
How can double helix of dna be separated?
Since it is bonded with H-bonds and it can be separated by heating or exposure to alkaline conditions
How does hybridization/renaturing of dna happen?
by gradual cooling or neutralizing the alkaline solution with a mild acid
Semiconservative
Fact that half of a newly made DNA is the old template
DNA replication is semiconservative
First in vitro DNA synthesis
-Arthur Kornberg
-used template DNA, DNA polymerase, 4 nucleotides
-if any of the nucleotides are missing DNA synthesis will stop
Experiment that prove semiconservative mechanism
- used isotopes to label old dna template and new dna made fromt that template
- grew bacterial in a medium containing ¹⁵N and allowed it to create 17 generations
- after took a sample from that and put them in a medium containg ¹⁴N allowed it to replicate thrice, collecting samples from all
- proved that dna replication is a combination of old and new dna strands
origin of replication
location on a dna molecule with a specific sequence that is recognized by enzymes involved in DNA replication
- in eukaryotes the origin of replication is nor that well defined
What is required for dna replication under in vitro conditions?
- dna template
- dna primer (short oligonucleotide)
- Mg⁺⁺
- dna polymerase
- dNTPs in a suitable buffer
dna replication in vivo
- dna replication starts at the origin of replication and proceeds biderectionally
- replication buble consists of two replication forks that grows wider as dna replication continues
dna in vivo replication- initiation
starts with relaxation of supercoiling at origin of rep. by topoisomerase
- resulting single-straded dna is stabilized by ssb proteins
- rna primer starts new dna synthesis
important enzymes and proteins
- topoisomerase
- ssb proteins
- primase
- dna polymerase iii (complex protein)
Primase (def)
makes an rna primer that provides a 3-OH group and which is needed for dna polymerase to work, also starts new dna synthesis
dna in vivo replication- elongation
- dna polymerase iii binds to dna template + rna primer region and starts to add complementary nucleotides
- dna synthesis always happen 5’ to 3’
because dna polymerase links only to 3-OH group - a leading strand is synthesized continuously
- dna is synthesized in okazaki fragments
Okazaki fragments
because the opposite strand is not fully open to continue dna synthesis from the 5’ to 3’ direction, dna is synthesized in small fragments called okazaki fragments
order in which dna enzymes work
- once dna strand is synthesized, dna polymeraze iii proofreads it makes sure errors are removed
- dna polymeraze i remove the dna primer and complete the dna strand
- once the small fragments are completed dna ligase joins the two ends of dna strands to complete dna replication
- dna gyrase facilitates the supercoiling of dna to compact the chromosomes into nucleosomes
Topoisomerase function
relaxes the supercoiled dna
Helicase
unwinds the double helix into a single-stranded dna
SSB
single-stranded dna binding protein. stabilizes the ssDNA
primase
synthesizes RNA primers
DNA polymerase I
erases RNA primers and fills in the gaps
DNA polymerase III Holoenzyme
DNA synthesis, proof reading, exonuclease activity and repair
DNA ligase
covalently joins free 3’ and 5’ ends of two DNA fragments through phosphodiester linkage
DNA gyrase
introduces supercoiling in DNA
Mismatch repair
- corrects error during dna replication
- dna polymease III
- these corrections use the mismatch repair mechanism by checking the complimentary bases pairing
Excision repair
- occurs after cell division, in the G1, S, G2 phase
- damage caused by carcinogens and mutagenic radiations altering bases or making pyrimidine dimers
- corrected by excising the damaged strands of DNA and making a new matching strand in its place
Telomere repair
- the enzyme telomerase extends the 3’ end of the telomere region and allows RNA primer to bind to its extended region protecting the telomere from being degraded
- occurs in young and actively growing cells, but also in cancer cells
