Exam 2: Lecture 1

Question 1

Semi-conservative model

Answer 1

• chains of the double helix would separate thereby providing templates for the production of a complementary strand of DNA
• two daughter helices would contain a parental template strand and a newly synthesized strand

Question 2

Background to Most Beautiful Experiment (nucleotides)

Answer 2

• bases of the nucleotides contain nitrogen atoms
• Bacterial cells will take up nitrogen atoms from the media
• nitrogen variants will be incorporated into the bases
• isotopes used by Meselson and Stahl were N14 and N15.
• since these isotopes have different molecular weights, the bases, nucleotides and synthesized DNA strands will be of different weights

Question 3

Most Beautiful Experiment

Answer 3

• grew bacteria for many generations in media containing the N15 isotope
• one subpopulation of cells were then transferred to media containing the N14 isotope for a single generation while another population of cells were grow in N14 containing media for several generations
• all three populations were then placed into an ultracentrifuge and pelleted through a Cesium Chloride (CsCl) gradient
• DNA from cells that grew only in N15 media pelleted to the bottom of the tube
• DNA from cells that had spent one generation in N14 containing media moved halfway through the CsCl gradient
• DNA from cells that spent several generations in N14 containing media pelleted in two locations: one halfway through tube, other at a higher location
• proved semi-conservative nature of DNA replication

Question 4

DNA Replication Initiation

Answer 4

• initaited at 100’s/1000’s of sequence specific positions called “origins of replication”
• as DNA is decondensed, these replication origins become accessible to several proteins collectively called pre-Replication Complex (preRC)

Question 5

Pre-Replication Complex (preRC)

Answer 5

-locally “melt” double helix creating replication bubbles

Question 6

Replication Bubble

Answer 6

• at each DNA synthesis is occurring in both directions
• over length of S phase, replication bubbles grow larger and will come in contact with each other as DNA synthesis proceedes through S phase

Question 7

Arthur Kornberg

Answer 7

• set about identifying enzyme(s) used to replicated chromosomal DNA.
• he and his research group published a series of papers reporting the discovery of DNA polymerase and the ability to reconstitute DNA replication in a cell-free extract system

Question 8

DNa Replication after Initiation

Answer 8

• after the initial melting of the double helix at the replication origins, DNA is further unwound by an enzyme called DNA helicases
• single stranded DNA strands are then bound by DNA polymerase which will read the two template strands in the 3 to 5 direction
• since two strands of double helix are found in an anti-parallel orientation this means that the two DNA polymerase complexes are moving along the two DNA strands in opposite directions
• one newly synthesized strand is called the leading strand and is made in one long continuous stretch
• other is called lagging strand and made in several short bursts of replecation
• new DNA strands are synthesized in the 5-3 direction
• means that the anti-parallel orientation of the DNA strands will be preserved in the daughter duplexes

Question 9

DNA Polymerase

Answer 9

• adds nucleotides to a growing polynucleotide chain one base at a time (above) the enzyme is considered to be functionally “processive
• means that a single polymerase molecule can add thousands of bases before falling off the template strand (right)
• can add nucleotides at a rate of nearly 1000 nucleotides per second
• means replication of genomes happens very rapidly

Question 10

Con of Rapid Rate of Adding Nucleotides

Answer 10

• potential for making errors (adding the wrong base)
• DNA polymerase has evolved the ability to proofread itself
• added base is checked and if an incorrect one has been added then the exonuclease activity of the protein will excise the incorrect base and add new base in its place
• process repeated until all newly added bases are judged to be correct

Question 11

Unwinding of DNA at Replication Fork

Answer 11

• after initial melting of the DNA double helix by the preRC proteins the replication bubble must continue to be unwound in order to expose the individual DNA strands to DNA polymerase
• constant unwinding of the DNA is accomplished through the enzymatic activity of DNA helicase
• helicases also used to unwind double helix during DNA repair, recombination and transcription

Question 12

Mutations in Helicase

Answer 12

• cause of several human disorders
• i.e. werener syndrome ages you rapidly and die by 50
• autosomal recessive
• incidence of 1 in 1,000,000

Question 13

Releiving Structral Tension Ahead of Replication Fork (DNA Topoisomerase/Gyrase)

Answer 13

• unwinding of double helix by DNA helicase puts physical strain ahead of replication fork
• tangling of duplex called positive supercoiling
• if tension not relieved, replication fork will stall as knots of DNA will become impossible to unwind
• DNA Topoisomerase will dissipate tangles by making double stranded break in DNA ahead of replication fork
• duplex allowed to untwist and topoisomerase will glue ends back together
• resulting DNA negatively supercoiled which is natural conformation of DNA double helix

Question 14

Autoimmune Diseases

Answer 14

• human body produces antibodies againsto topoisomerase I and II
• once bound by antibodies, acitivity of both types of topoisomerases are either completely blocked or severely reduced
• loss of topoisomerase activity has been implicated in CREST Syndrome, systemic lupis erythematosus, and chronic prolifeative kidney diseases

Question 15

Protecting DNA Strands During Replication

Answer 15

• DNA helicase unwinds the double helix the two polynucleotide chains will, for a short time, be single stranded
• in this state the bases of the two DNA strands could prematurely pair thereby undoing the activity of the helicase
• if localized regions in which the two strands rejoin replication can pause which is associated with replication slippage which is the expansion of DNA segments
• been suggested that replication pausing and slippage may contribute to the incorrect addition of CGG and CTG sequences during replication
• addition of Single Stranded Binding Protein (SSBP) can prevent the base pairing of single DNA strands and thus prevent replication pausing and slippage
• postulated that reduction sin SSBP activity could be contributing factor to the triplet expansion disorders.

Question 16

Mutations in SSBP

Answer 16

• number of mitochondrial diseases attributed to this because mitochondria have a small genome that needs to be replicated
• withoutSSBP replication pausing can occur leading to the accumulation of mutations within the mitochondrial genome
• disorders have many neurological phenotypes such as migraine headaches, epilepsy and Parkinsonism