BIOC520: Biological Chemistry			
L-41/43: DNA, DNA REPLICATION, DNA REPAIR

Objectives:
1.	To examine the basic structure and properties of DNA and how it is
packaged in the eukaryotic nucleus
2.	To understand the process of DNA replication
3.	To examine the pathways whereby damaged DNA can be repaired


I.	Overview of genetic information flow


II.	Structure of DNA (Fig. 11.5-11.8  Marks)
  A.	DNA is a polymer of deoxyribonucleotide monophosphates
    1.	bases are adenine, guanine, cytosine, and thymine
    2.	linkage is through phosphodiester bonds
    3.	5' and 3' ends
    4.	every DNA has a specific sequence of nucleotides--its primary
structure--genetic information is stored in the primary structure of DNA

  B.	most DNA in a cell exists as the Watson-Crick double helix which
is known as B form DNA (Figs. 11.8-11.14)
Major features: right handed helix; bases on inside and sugar phosphate
backbone on outside; base pairs are formed through hydrogen bonding; A
pairs with T; G pairs with C; bases are perpendicular to the helical axis
stacked on top of each other and interacting through hydrophobic
interactions and van der Waals interactions; 3.4 A per base pair; ~10 base
pairs per helical turn; strands are antiparallel; strands are
complementary

A - DNA
Z - DNA

  C.	DNA supercoiling (Fig. 28.33, 28.37, 28.41, 28.43, 28.44  Voet)
    1.	a supercoil is when the double-helix twists around itself
    2.	supercoils can be positive or negative but natural DNAs exist in
the negative supercoiled form
    3.	DNA can be supercoiled if it is circular or if is linear and has
fixed ends
    4.	supercoiled DNA is more compact than relaxed DNA
    5.	negatively supercoiled DNA molecules are easier to unwind than
relaxed molecules
--DNA unwinding is required for replication and transcription

Topoisomerases are enzymes that catalyze changes in DNA supercoiling (Fig.
28.41, 28.43, 28.44  Voet)
    1.	Type I topoisomerases function by breaking a phosphodiester bond
of one strand, passing the other strand through the break and resealing
the break--they can only remove supercoils
    2.	Type II topoisomerases function by breaking both strands and
passing a double strand region through the break before resealing the
break--require ATP
    3.	topoisomerases are targets of numerous chemotherapeutic drugs:
adriamycin, VP16 (tenoposide), VM26 (etoposide), camptothecin

  D.	Nucleases--enzymes that catalyze hydrolysis of phosphodiester
bonds in nucleic acids
    1.	exonucleases cleave terminal nucleotides from either the 5' or 3'
end of a polynucleotide
    2.	endonucleases cleave in the interior of nucleic acid
molecule--restriction enzymes are endonucleases that cleave at specific
sequences of DNA

  E.	Denaturation and renaturation of DNA (Fig. 11.15-16)
    1.	denaturation is the conversion of the double stranded form of DNA
into single stranded form
	a.	DNA can be denatured by heat or alkaline treatment
	b.	The temperature at which half the DNA is unwound is defined as the
melting temperature (Tm)
--Tm is dependent on the GC content of the DNA, on the solvent, and on the
ionic strength
	c.	hyperchromic effect
    2.	renaturation--under proper conditions, complementary
single-stranded nucleic acids can renature into a double-stranded form
    3.	denaturation/renaturation is the basis of hybridization
experiments--this type of analysis is central to recombinant DNA
technology and gene manipulation 

  F. 	Packaging of DNA in eukaryotes (Fig. 11.21-24)
    1.	DNA is packaged in the nucleus as a nucleoprotein complex called
chromatin
    2.	Levels of chromatin packaging:
	a.	nucleosomes: ~200 bp DNA and histones
	  1.	nucleosome core particles consist of ~140 bp of DNA
wrapped around a protein octamer consisting of 2 subunits each of histones
H2A, H2B, H3, and H4
	  2.	linker DNA is the DNA between two core particles
	  3.	histone H1 binds to the linker DNA and the core particle
	b.	30 nm fiber: nucleosomes are wound into a solenoid-like
structure--requires histone H1 binding to every linker DNA
	c.	higher levels of chromatin packaging
  3.	chromatin is the template for replication and transcription and
the substrate for DNA repair and recombination


II.	DNA Replication (Fig. 12.1-12.5  Marks)
  A.	DNA replication is semiconservative
  B.	DNA replication is (usually) bidirectional
  C.	Chain growth occurs by addition of deoxynucleotidyl monomers to
the end of a DNA chain
    1.	DNA  is always synthesized in the 5' to 3' direction
    2.	This reaction is carried out by DNA polymerases (Table 12.1)
	a.	general requirements of DNA polymerases
	  1.	deoxynucleotide triphosphates (dATP, dGTP, dCTP, TTP)
	  2.	DNA template
	  3.	a primer chain with a free 3'-OH--note that DNA
polymerases cannot
start with a single                         nucleotide
  D.	At a replication fork one of the new strands is synthesized in a
continuous manner (the leading strand) and the other is synthesized in a
discontinuous manner (the lagging strand)
      
  E.	Molecular events of DNA replication  (E. coli chromosomal
replication)    (Fig. 31.15, 31.23, 31.22  Voet)
    1.	Initiation
	a.	an initiator protein (dnaA protein in E. coli) binds to an origin
of replication and "melts" a short DNA sequence
--an origin is a unique site on the chromosome where replication begins,
it consists of binding sites for the initiator protein and a flanking
AT-rich sequence
	b.	helicase (dnaB/dnaC complex) binds to melted region and further
unwinds parental strands
	c.	SSB (single-strand binding protein) binds to unwound region to
prevent reannealing and stabilize the single-stranded form at the
replication fork
	d.	an RNA primer is synthesized by primase (primase is part of a
multi-protein complex called the primosome)
	e.	DNA polymerase III (holoenzyme) is assembled
    2.	Elongation
	a.	DNA polymerase III extends RNA primers
	b.	helicase continues to unwind parental DNA strands ahead of
polymerase 
		1.	the leading strand continues uninterrupted (DNA pol III has high
processivity)
		2.	on the lagging strand, approximately 1000 bp are replicated before
primase must synthesize a new RNA primer to be elongated by DNA pol III
--Okazaki fragments
              	3.	DNA polymerase I (in E. coli)
removes RNA primer (5'-3' exonuclease activity) and fills in the gap (DNA
polymerase activity)
		4.	DNA ligase seals the nicks--E. coli DNA ligase requires NAD+
    3.	Termination of replication takes place within a region of the
circular E. coli chromosome called ter
  F.	The replisome is a large multiprotein "machine" that is thought to
replicate both the leading and lagging strand simultaneously
  G.	Proofreading: Pol III (and Pol I) has 3--5' exonuclease activity
--if incorrect  base is inserted it can back up 1 nucleotide and then
continue polymerization
  H.	DNA replication in eukaryotes (Fig. 12.7-12.9, Table 12.2)
    1.	enzymatically the mechanism is basically the same as in
prokaryotes
    2.	each chromosome has multiple origins  
    3.	DNA is replicated only during S phase of the cell cycle and only
once during each cell cycle
    4.	the substrate (template) for replication is chromatin
    5.	a special enzyme called telomerase is necessary for replicating
the ends of linear chromosomes
	a.	the ends of eukaryotic chromosomes are called telomeres and are
made up of short repetitive sequences (Fig. 31.34  Voet)
	b.	telomerase is an enzyme that contains both protein and RNA
components
	c.	the RNA component is used as a template to synthesize new telomere
repeats
	d.	telomerase and cancer


III.	DNA REPAIR
  A.	DNA damage caused by ultraviolet light
    1.	cyclobutane-type pyrimidine dimer is the major photoproduct formed
(Fig. 12.11)
    2.	a second product, the 6-4 photoproduct, is formed in about 10 % of
UV induced pyrimidine dimers
    3.	the cyclobutane type dimer can be reversed by a process called
photoreactivation
	a.	this is carried out by an enzyme called DNA photolyase
(photoreactivating enzyme)
	b.	importance of the photolyase enzyme in humans is questionable
    4.	DNA photoproducts can also be repaired by excision repair (Fig.
12.12)

  B.	Spontaneous deamination of cytosine (Fig. 12.13)
    1.	deamination of cytosine is common and results in the conversion of
cytosine to uracil
    2.	can be repaired by excision repair process
	1.	uracil-DNA glycosylase hydrolyzes N-glycosidic bond to remove
uracil base
	2.	AP endonuclease removes deoxyribose-phosphate
	3.	gap is extended by exonuclease 
	4.	gap is filled by DNA polymerase I and nick is sealed by DNA ligase 
       
  C.	depurination--pathway is similar to that above except that
excision repair begins with AP endonuclease
  D.	DNA damaged by alkylating agents
    1.	some simple alkylating agents:






    2.	examples of products of alkylating agents:







    3.	many of these products can be repaired by excision repair that is
initiated by specific glycosylases
    4.	some damage resulting from methylation can be reversed by
methyltransferases
	a.	O6-methylguanine-DNA methyltransferase
  E.	Mismatch repair (Fig. 12.14)
    1.	mismatches can occur when DNA polymerase inserts the wrong
nucleotide during replication
    2.	mismatch repair is "coupled" to replication   
    3.	How do the mismatch repair enzymes distinguish which base is
incorrect?
	a.	parental DNA is methylated--in E. coli an enzyme called Dam
methylase methylates the C in both strands at the sequence GATC
	b.	immediately after replication only the parental strands are
methylated (the DNA is hemimethylated)
    4.	the defective gene in one form of hereditary colon cancer was
recently found to be the human homologue of mutS
  F.	Recombinational repair (Fig. 31.41)
    1.	occurs during DNA replication
    2.	major steps:
	a.	DNA polymerase skips over damaged DNA leaving a gap opposite the
lesion
	b.	the undamaged parental strand recombines into the gap (this is
facilitated by recA protein in E. coli)
	c.	the new gap in the parental strand is filled by DNA polymerase and
ligase
  G. 	Genetic Defects in DNA repair and human disease
    1.	Xeroderma pigmentosum is an inherited disease that is
characterized by severe photosensitivity and a very high incidence of skin
cancers. It is due to defective excision repair.
    2.	Bloom's syndrome.
    3.	Cockayne's syndrome
    4.	Fanconi's anemia
    5.	Ataxia telangiectasia