DNA # 17-20 Flashcards
What kind of bond is made between the 5’ oxygen of one deoxyribose to the 3’ oxygen of the next deoxyribose
phosphodiester bond
Are hydrogen bonds covalent bonds?
no! they are reversible (they can dissociate
aromaitc rings of bp form ……interactions?
favorable stacking–> stabilizes double helix
Most DNA exists in a coformation named A or B?
B form
Is the double stranded helix right handed or left handed?
Right handed
how many base pairs are there per complete turn of the helix
10 base pairs
What is the major and minor groove
Major groove has a lot of base pairs exposed , allowing for specific recognitino by proteins that bind to DNA
What is the extensive negative charge on the backbone neutralized by?
Salts uch as Na+, K, Ca, Mg or by organic amines
What organic amines can neutralize negative charge on backbone of DNA? (4)
Putrescine
cadaverine
spermine
spermidine
what is the avg length of helix chromosome?
5 cm, and 46 of them have to fit into each nucleus
What was Rosalind Franklin’s contribution of the DNA structure?
key contirbutoer to solving structure
named te A and B form of DNA
argued that phosphate backbone was on the outside, based on the amt of water that B form abosrbs, which requires a fully hydrated state
What kind of information was gathered from Photo 51
- crystal packing was in C2 symmetry group (centered with 2 fold axis of symmter, if turned 180 degrees, will ge tsame image b/c antiparallel)
- X shaped pattern indicated that it was helix
- spacing in the diffraction pattern indiacted teh diamter and the vertical rise and pitch of helix
What is the complete set of DNA for an organism called?
genome
Much of the DNA is orgnaized into ?
genes – >code for protein
how many bp in an entire genome of a species
3.5 billions
how many bp in a human genome? How many chromosomes is it packaged into?
3..2 biollion bp long
packaged into 23 or 24 different chromosomes, 2 copies of each chromosome per cell (except for germ cells and specialized cells like RBC)
what happens if you add choltricine to chromosomes?
it arrests chroosomes at condensed stage–> allow better visualization for karyotyping
What is GIEMSA Stain?
helps with karyotyping
Stain darkens regions of the chromosome rich in AT pairs
What do red knobs on chromosome 13, 14, 15, 21, 22 represent (pg 13 of notes)
genes that code for large ribosomal RNAs
What three elements are required for chromosome to undergo replication and mitosis?
- Each chromosome has many REPLICATION ORIGINS. These are points where duplication fo DNA can begin
- Each chromosome needs two telomeres, one at each end of chromosome. Contain repeated nucleotide sequences. Serve to cap ends, os that they dont look frayed ends in need of repair
- Each chromosome has a single CENTROERE. specialized sequence that allows for sepeartion of duplicated chromosomes at MITOSIS. Also keeps two chromosomes together prior to mitosis
HOw many base pairs does chromosome 22 have?
48 million
How long will chromosome be when fully extended? How long is it during mitosis?
1.5 cm long
during mitosis, condensed to 2 um in lenght, shorter by factor of 10,000
How is tight packaging accomplished?
Histones
How many histones does each chromosome have ~
over a millino histones ass. wtih it
What is chromatin
Combination of DNA + ass proteins
How thick is a chromatin fiber?
30 nm thick
what happens when 30 nm thick chromatin fiber is subjected to low salt conditions
partially unfolds producing “BEADS ON A STRING”
how compacte when DNA is wrapped aroudn preotein cores?
3 fold compaction
what hapepns when BEADS ON A STRING IS treated with nuclease? (enzyme that digests DNA)
what are resulting particles called
connecting strands are digested away, while DNA wrapped aroudn prtoein core is protected
resulting particles are called NUCLEOSOMES
What are nucleosomes? Composition?
how many Bp?
How many proteins?
Names of types of proteins
What are Histones
Each consists of DNA (147 bp in length) and
8 proteins of histone
(2 copeis each of histones H2A, H2B, H3, and H4
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes.[1][2] They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation.
How many turns does DNA make around the histone core ina nucleosome?
1.7 turns
How many base pairs in linker DNA
varies in length from a few bp to ~80
What kind of amino acids (charge) compose histones? Why?
Positively charged amino acids (lys and arg) facilitate interaction with Negatively charged phosphate backbone of DNA
What kind of organisms possess histones/
Euk and Arcaea ( not bacteria)
most ighly conserved proteins known
What side (N or C) are tails of histones?
N-terminal end of protein. Not as tightly packed
tails are subject to enzymatic modification that can regualte chromatin structure and gene expression
Where does Histone H1 bind?
binds at point where NDA exits teh nucleosome
Its N terminal and C terminal tasla re beleived to cause nucleosome to pack even mroe densely
Why do you condense chromatin into a chromosome?
Mitosis?
How do you unpack DNA to Make protein?
1st method utilize CHROMATIN REMODELING COMPLEXES
What are chormatin-remodeling complexes?
how do they work?
bnd to DNA and to histones
Powered by ATP hydrolysis , slide along the DNA, moving or removing the histones and decondensing local regions of DNA
During mitosis, thsees complexes are inactive
what are ways to restructure chormatin (if you want to maybe use the DNA for protein synthesis
- DNA unpakcing with Chromatin remodeling complexes
2. Histone modifications by adding
HOw are tails of nucleosme core hisotnes enzymatically modified?
Acetylation
Methylation
Phosphorylaion
Acetylation
usually occurs on (+) lysine chains
Acetylation neutralizes lysine charge
Methylation
can occur on LYSINE (+) or ARGININE (+)
methylation does not neutralize the positive charge but it spreads the charge over a larger number of atoms (decrease attraction of histone core to (-) DNA)
Phosphorylation
Occurs on Hydroxyl groups of serine or threionine
adds a negative charge (repes)
Descirbe Histone H3
composed of 135 amino acids, which make up globular protein
What is a histone code?
different modification patterns
Where does heterochromatin reside in cell?
WHAT DOES IT CONTAIN
usually accuulates at the periphery of nucleus
INcludes a lot of non-gene-coding DNA , including CENTROMERE AND TELMOERES
Not active during interphase b/c contains protions that arent neeeded fo r synthesis
What happens when chromosomal rearrangement takes place andis inserted into heterochromatin
gene may be poorly expressed or completely suppresed
What is a Barr Body?
When does it occur?
When one copy of X chromosome is permanently condensed to heterochromatin and is not expressed (it is silenced)
This happens when it is 1,000 cell
Inactivation patterns are inherited.
What cells are genes codign for skeletal muscle present in?
In skeletal muscle cells,
should be silenced in other cell types
How are DNA strands labeled?
5’ –> 3’ based on position of fre hydorxyl (OH) group of deoxyribose
What enzyme is responsible for adding new nucleotides to the growing strands
adds nucleoside-5-triphosphate to free 3’ hydorxyl of chain
Chain is built 5’ –> 3’ direction
where does energy come from to power the synthetic step?
energy from hydorlysis of nuceoside-triphosphate
pyrophoshate is released in the process
what is an exonuclease?
removes single nucleotide, proofreading
What is a Exonuclease 3’5’? funciton?
DNA Polymerase
Proofreading (removes newly added nucleotide that is not correclty based paired
How many DNA polymerases do Prokaryotes have?
4, 3 main ones summarized aboe
What is DNA POL I (in ecoli?)
Involved with initialtino of DNA syntehsis when organism replicates. REMOVES PRIMER!!!!
requires a 3’ priamre and template strnd to carry out DNA synthesis
after it adds 20 nucleotides, it falls off and DNA POL III takes over DNA synthesis
What is Pol II?
What happens if you delete it?
invovled with DNA repair
if deleted, Pol II doesnt interefere with gorwht or replication
aslo has a 3-5’ exonculease activity
POL III
Main Polymerase!
involved in replicatino of DNA
HIGH PROCESSIVITY ( add 100,000 nucleotides before it falls off)
Pol IV and V
replicate DNA without proofreading
invoved with repair, may replicate though dmaged DNA
what direction does DNA synthesis go
always 5’–>3’
How many DNA Pol does euk have
8-14ish
Pol alpha
Intiiation of DNA synth
forms complex with DAN primase that makes the RNA primaer
after alph adds ~20 nucleotides, it falls off, and pol delta or epsiolon take over DNA synt
No 3-5 exonuclease activit to proofread or 5-3 exonculease activity
Pol Beta
DNA repair (base excision repair)
Pol gamma
replicatino of mitochondrial DNA
has 3-5’ exonculease activity
Pol gamma and pol epsilon
replicate nuclear DNA with high processivity
MAIN polymerases for DNA replciation
what removs primers in euk?
what fills in gap?
RNAse H,
gap filled by Pol alpha
Pol alpha also does repairs
What is the error rate of DNA polymerase?
rate of 1/10,000,000
through proofreading activity
what apr tof enzyme cleaves off an incorrectly added nucleotide
Nuclase domain of enzyme
what is occuring ont eh P site? what about E site?
P site –> domain wher Polyermization is carreid out
E site- where proofreading (error-checking) takes place
what kind of bonds are holding together two complementary strands
H-bonds
each bond is reversible/non-covalent assicoiation with only a few kcal/mole of binding energy
sum of energies over whole lenght of DNA (combined with favorable ENTROPY of the assembly) makes it diffilut to separate the two strands completely
need temp near bp of water to separate strangds completely
what are REPLICATION ORIGINS?
what do intitiator proteins attack?
speciifc reigons of DNA that attract intiaitor proteins that can sepearte teh two strands
strategy incldue breaking apprt a few bp at at time (divide and conquer) and a high proprotion of AT pairs in teh replication origins (attacks the weaker A-T pairs rather than stronger GC pairs)
What is an Origin Recogntion Complex (ORC)
mult-subunit complex that first binds to specific DNA sequences that are replication origins
ORCs deets
- speciic replication origin sequences attract the necessary machinery regulated by cell
- process requires energy ATP
- machinery include helicases,
What is OriC?
AT rich region in E.coli
bacteria usually have only single replicatino origin
Eukarytoes- how many replciation origins?
multipe!
about 10,000 in humans!
what direction does replication occur?
how fast does replication fork move in pro and euk
bidirectional; nucleotides added 5–>3
fork moves 1000 nuclteotides per second in bacteria
100 per second in euk b/c of more complex chormatin structure
What sid ecan DNAY Polymerase add nucleotides to (5’ or 3’)?
3’-end of an existing nucleic acid chain
How does lagging stand get started ?
RNA primer is constrcuted on teh exposed ss DNA by enzyme called DNA primase
DNA primase is an RNA polymerase
What is DNA PRIMASE?
A type of RNA polymerase
uses ss DNA as template and constructs RNA strand ofa bout 10 nucleotides in length
3’ end of this RNA strand then serves as teh “starter for DNA polymerase to work on
DNA polymerase keeps going until it runs into a notehr RNA primer
What three enzymes are needed to finsih the job of syntehsis after RNA primer is added by a RNA polymerase (called DNA Primase)
- Nuclease-removes RNA primer
- Repair DNA polymerase filsl int eh gap- using 3 ‘ end of Okazaki fragment as its primer
- DNA ligase joins the 5’-end of one frgrament with the 3’ end of the other
Nuclease, repair DNA polyermase, DNA ligase
DOes DNA primase have proofreading capability?
NO! b/c it is RNA!
but it is advantagious to use RNA instead of DNA b/c replacement of RNA will be replaced!!!!
what is Helicase?
where does it get energy?
unwinds ds DNA usign energy from ATP hydrolysis to its tranit along the cahin
What is problem with lagging strand during undwinding? What protein inhibits this problem from occuring?
lagging strang ust be prevented form re-foring bp.
this is done using multiple copies of ss binding protein
SINGLE STRANDED BINDING PROTEIN - prevesnt lagging strand form re-foring bp
what is a Sliding clamp protein? Function?
WHAT PUTS CLAMP PROTEIN in its place?
Keeps DNA Polymerase firmly attached to template
slidingn clamp protein is put in place by another ATp powered preotin called CLAMMP LOADER
what is Helicase?
where does it get energy?
unwinds ds DNA usign energy from ATP hydrolysis to its tranit along the cahin
What is problem with lagging strand during undwinding? What protein inhibits this problem from occuring?
lagging strang ust be prevented form re-foring bp.
this is done using multiple copies of ss binding protein
SINGLE STRANDED BINDING PROTEIN - prevesnt lagging strand form re-foring bp
what is a Sliding clamp protein? Function?
WHAT PUTS CLAMP PROTEIN in its place?
Keeps DNA Polymerase firmly attached to template
slidingn clamp protein is put in place by another ATp powered preotin called CLAMMP LOADER
