Bio 121 Exam 4 Set 2
What is ORC?
ORC: origin recognition complex -binds to ARS element Eukaryotes -makes sure DNA is only replicated once by recruiting licensing factors
Why is there an RNA polymerase in every replisome(replication complex)?
RNA polymerase is called primase -makes a short piece of RNA called a primer of DNA plymerase to add to
What does it mean to say DNA replication is semi conservative?
a newly synthesized DNA molecule consists of one "old" stand and one "new" stand therefore semi conservative
What is a plasmid and what does every plasmid have?
plasmid: small circular DNA molecules in bacteria which encode proteins that inactivate certain antibioticsall plasmids contain origins of replication which tell the cell how to replicate the plasmid and how many copies to make
What do SSB proteins do and why are they necessary?
single stranded binding proteins stabilize single stranded regions
What is an origin of replication?
specific sequence of DNA nucleotides where DNA is opened and untwisted by helicase; recognized by initiator proteins
What does gyrase do and why is it necessary?
relieves tension from untwisting of DNA relieves supercoiling
Why are there so many proteins in a DNA replication complex?
replication involves solving many problems each problem is solved by a different protein (enzyme)
What is a restriction enzyme and what is their purpose in nature?
restriction enzymes cut DNA at specific sequences called restriction sites. they are produced by bacteria as a defense against viruses
Why is there a ribonuclease in every replisome?
ribonuclease removes RNA primer (will be replaced with DNA nucleotides)
How did Watson and Crick propose that DNA was replicated?
semi conservative method: each "old" strand used as a template for the "new" stands
You have identified a new virus with a single-stranded DNA genome. After centrifuging DNA from infected cells in CsCl you ovtain two bands of different density. One is 20% A, 30% C, 30% G, 20% T. the other is 20% A, 20% C, 30% G, 30% T. Which band is viral DNA?
the second one because it is not equal
How do we fingerprint human DNA?
we use PCR to detect which version we have for each of the 13COD15 microsatellite loci used by the FBI chromosomes #16 for ALU sequence chromosomes #4 for microsatellites
What is the telomerase theory of aging?
without the action of telomerase chromosome ends shorten -normal cells undergo a specified number of cell division correlated with length of telomere -telomerase is active in embryonic cells and cells of children, but low in adult somatic sells (except cells like lymphocytes which divide as part of their function) -these is a relationship between cell senescence (aging) and telomere length; "mature" cells don't make telomerase
What is the loop model for replicating DNA?
-DNA is "fed" through a replisome -lagging strand is looked out -both DNA polymerases move in the same direction
What are telomeres?
-DNA sequences at the ends of euk. chromosomes -in humans, consist of 250-7000 repeats of CCCTAA -protect the ends of chromosomes since special proteins bind the telomeres with each replication, a telomere is removed
What is nucleotide excision repair?
-damaged region of DNA is removed and then replaced by DNA synthesis -involves: a. recognition of damage b. removal of damaged region c. resynthesis of removed segment using undamaged strand as template (DNA polymerase I or II) DNA ligase seals the "nick" in the DNA back bone
Why is replicating the ends of linear DNA molecules a problem?
-directionality of polymerases plus need for a primer are the problem -no problem with leading strand; but lagging strand only replicated to last primer; leading strand is therefor longer than lagging strand -each mitosis, telomeres get 200 base pairs shorter on each end
What causes Xeroderma pigmentosum?
-disorder resulting from inability of cells to correct DNA damage due to UV - 8 genes are involved in DNA repair caused by UV -individual is ultra-sensitive to UV
How do eukaryotic cells ensure that each origin of replication only fires once per cell cycle?
-each ARS is replicated as a discrete unit -licensing factors ensure ARS is replicated once per S phase; these fall off (as do actiration factors) once replication starts and are thrown out of nucleus. they don't reattach until after mitosis (late telophase)
What is telomerase, and what does it do?
-enzyme that replaces missing bases-uses an internal RNA as a template (not the DNA itself) -use of internal RNA allows short segments of DNA to be made -these segments are repeated nucleotide sequences complementary to the RNA of the enzyme; legging strand is completed by normal DNA synthesis
What is a mismatch repair?
-fix bases that aren't paired properly -process: 1. enzyme find mismatched bases 2. replace base on new strand using old strand as template (cell assumes "old" strand has correct information) ("old" strand is methylated)
What is base excision repair?
-fixes altered and missing bases -process: 1. enzyme find and remove defective bases 2. DNA polymerase (beta) replaces missing base(s) 3. DNA ligase seals nick in backbone nucleotide excision repair ~30 nucleotides VS base excision repair-1-3 nucleotides
How did Meselson and Stahl prove that Watson and Crick were correct?
-labeled DNA with heavy nitrogen 15N -parental DNA N15N15 -transferred t medium containing N14 -F1 -N15N14 and N14N15 -F2-N15N14, N14N14, N14N14, and N14N15
How did Beadle and Tatum show that genes are instructions for making enzymes?
-one gene-one enzyme hypothesis-deliberately destroyed genes, then looked for heritable defects in enzymes -mutants unable to grow on minimal media -identified missing nutrient by adding specific chemicals, one at a time -each allele coded for a different enzyme in pathway used for arginine production A-->B-->C-->D-->arginine genes store information for the production of proteins. information is stored in the sequence of nucleotides of the gene
What is DNA proof-reading and how does it work?
-only correct DNA can exit replication process -proof reading is the most important aspect of replicating DNA correctly -if bases are mis-paired, process backs up and "tries again" -most DNA mistakes are corrected this way
What is the role of the "sliding clamp"?
-subunit of DNA polyerase III (enzyme that adds DNA nucleotides, making "new stand") -forms a ring around DNA template, holding the enzyme in place
What is ARS?
ARS: autonomously replicated sequence -section of DNA replicated at each origin of replication -average 100,000 base pairs in length -each zone replicated as a unit -increases speed of replication
What is a photolyase?
An enzyme that repairs dimer using energy from visible (blue) light
Why do defective BRCA1 or BRCA2 genes increase the rick of cancer?
BRCA 1 and BRCA 2 work with protein RAD51 to repair double-strand breaks. If these are defective, DNA damage won't be corrected -if both BRCA1 are defective 80% cancer risk -if both BRCA2 are defective 70% cancer risk
What does telomerase have to do with cancer?
Cancer cells slow activation of telomerase, which allows chromosomes to maintain their length. This is one aspect of cancer that allows for cell division that continues indefinitely -Telomerase is a symptom, not a cause, of cancer. Serum telomerase is used to diagnose cancer
Why are there leading and lagging stands at every DNA replication fork?
DNA polmerase an only add in 5' to 3' direction; antiparallel stands are copied by different mechanisms
Why do cells need RNA primer to start DNA replication?
DNA polymerase adds DNA nucleotides to an RNA primer
How does UV damage DNA, and how is this damage repaired?
UV causes formation of thymine (pyrimidine) dimers adjacent thymine bases link together -photorepair occurs: enzyme called photolyase, absorbs visible light and uses the energy to cleave thymine dimer -note: sunlight in UV range causes damage. sunlight in Visible range used to repair damage
What is a mutagen?
agents (radiation, chemicals) that damage DNA DNA sequence is incorrect (A,C,T,G)
Why do eukaryotic cells have multiple origins of replication on every chromosome?
allows DNA to be replicated quickly (more DNA than in prokaryotes)
Why is DNA ligase associated with each DNA replication complex?
attaches <--> anneal Okazaki fragments together by creating phosphodiester bonds
How do bacteria protect their DNA from being destroyed by their own restriction enzyme?
bacteria cell shave a restriction endonuclease/methylase pair that protects bacterial recognition sequencesbacterial DNA is methylated which protects it from restriction enzymes
How do cells know where to start replicating DNA?
begins at origin of replication; initiator proteins bind here and recruit replication complex tell DNA polymerase where to start
Why is the loop model necessary?
both leading and lagging strands are replicated in same direction
Why did you add calcium chloride to your bacteria and why did you heat shock them?
calcium chloride loosens up cell wall so plamid can get through heat shock allows plasmid to enter cytoplasm through cell membrane
How do X-rays damage DNA?
causes double-strand breaks
Why did you spread your bacteria on plates containing amplicillin?
cells which took up plasmid should be able to grow on medium containing amplicillin; bacteria that did not take up plasmid will be killed by amplicillin this is how transformants (these bacteria have new DNA (the plasmid) are slected
What is the cause of Hereditary Nonpolyposis Colorectal Cancer?
defective repair of mismatch mutations "if you can't fix DNA, you have a problem"
What evidence supports the hypothesis that each gene encodes a protein?
destroy gene, no enzyme (a gene contains instruction for the production of a protein) product of a gene is a protein
What is restriction mapping?
determine the relative positions of restriction sites
Why did Gerrod conclude that some diseases have a genetic basis?
disease was "in the family"; inherited
What is an Okazaki fragment?
fragment of DNA on lagging stand
What holds the new base in place until DNA polymerase has catalyzed the phosphodiester bond?
hydrogen bonds between complementary bases
Where does the energy needed to catalyze the phosphodiester bond come from
hydrolysis of two PO4 molecules use ATP ATP---->ADP+P+energy
Which proteins bind to the origin of replication?
initiator proteins-recognize and bind to origin of replication/recruit replication complex (ex. helicase, gyrose) -forms a complex that opens the helix and separates strands -specifically: --bacteria with initiator proteins --eukaryotes use ORC: origin recognition complexes these proteins bind to specific DNA sequences to begin replication
Why do we say that DNA replication is semi-discontinuous?
leading stand is copied continuously towards replication fork lagging stand is made in fragments away from replication fork (discontinuous)
How does the cell determine which base to add next?
only one base fits use complementary stands as template
What are the problems which must be solved in order to replicate DNA?
open DNA keep it open prime copy anneal (join segments of DNA together)
What does helicase do?
opens and untwist DNA "melts" DNA
