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BL414 Genetics Spring 2006
Lecture 10 Outline
February 27, 2006
Chapter 6: DNA replication and recombination
It is necessary for an organism or cell to replicate its genome during mitosis and
meiosis, and this is done by the process of DNA replication.
6.2 In DNA replication, the two complementary strands of the double helix of
DNA are separated and a new complementary strand is formed for each parent
strand, creating two new double helices, identical in sequence to each other and
to the parent helix. This is called semiconservative replication because each
daughter cell is composed of half parental material and half new material.
The steps of DNA replication are initiation, elongation and proofreading.
Initiation: a short complementary RNA fragment called a primer base pairs to
the parent strand and from this, new dNTP’s are added to the 3’ end.
Elongation: more complementary dNTP’s are added to the nascent 3’ end of the
DNA daughter strand, always in the 5’ 3’ direction
The leading strand is synthesized continuously, because it points in the direction
in which the helix is opening up during replication. The other strand, called the
lagging strand, is synthesized in a discontinuous fashion because it must be
elongated in the 5’ 3’ direction, and therefore is going in the reverse direction
of the unwinding double helix (cf. Fig. 6.13 and 6.20). The fragments of DNA
discontinuously synthesized on the lagging strand are called precursor
fragments or Okazaki fragments. In bacteria the fragments are 1000-2000 base
pairs and in eukaryotes they are only 100-200 base pairs in length.
Incorrect nucleotides are incorporated by the replication machinery at a rate of 1
in 100,000 nucleotides, which would result in about 60,000 base pair changes in
every cell cycle. There is a proofreading step in DNA replication that prevents
the generation of this many mutations.
Circular DNA is found in bacterial and some viruses and is replicated by 
replication or rolling-circle replication.
In  replication, DNA replication begins at an origin of replication and usually
proceeds in two directions (bidirectional) outward from the origin, so there are
two replication forks at which DNA replication is occurring.
In rolling-circle replication, one of the DNA strands is nicked by a nuclease,
leaving a 5’-phosphate group and a 3’ hydroxyl group. New nucleotides are
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added to the 3’OH, complementary to the opposite DNA strand. Elongation
proceeds around the circular DNA and may continue around the complete circle
multiple times. The nicked strand is also replicated in a discontinuous fashion as
it is displaced from the rolling circle.
In eukaryotic chromosomes, DNA replication initiates at multiple origins of
replication on chromosomes and elongates bidirectionally along the linear
chromosome.
6.3-6.6 Besides the DNA synthesis machinery many other proteins are involved
in DNA replication.
 Helicases are necessary to unwind the DNA. These use ATP to drive the
unwinding reaction.
 Single-stranded binding proteins (SSP’s) are necessary to stabilize the
unwound strands of DNA.
 Gyrase relieves the rotational stress on DNA caused by the unwinding of
DNA at the replication forks. Gyrase does this by breaking the dsDNA,
swiveling it around and rejoining the broken double strands. Gyrase is a
topoisomerase.
 Primase, which functions in bacterial DNA replication, is a special RNA
polymerase that synthesizes the short RNA molecule primer necessary for
initiation of DNA replication. A similar job is carried out in eukaryotes by
polymerase , a component of the primosome, which synthesizes 12
nucleotides of RNA connected to about 20 nucleotides of DNA, the RNADNA primer used in eukaryotic DNA replication.
 DNA polymerase , a component of the DNA polymerase complex, adds
dNTP’s to the 3’-OH end of the primer and continues adding more
nucleotides to the 3’ end of the new daughter strand.
 DNA synthesis always proceeds in the 5’ to 3’ direction. (cf. Fig.
6.18)
 The DNA polymerase complex also contains a 3’-to-5’ exonuclease that
corrects errors of incorporation (incorrect nucleotides). A mismatched
base will have a different structure than a correct base pair and the
mismatched structure will be recognized by the 3’-to-5’ exonuclease,
which will cleave the DNA backbone phosphodiester bond at that point.
Cleavage of the DNA at the mismatch position allows a new, correct
dNTP to come in and be incorporated. This is the proofreading function
of DNA replication.
 The RNA portions of the primers left behind must be removed in order
complete synthesis of the daughter DNA molecule. In bacteria, a 5’-to-3’
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exonuclease activity, Pol I, removes the RNA nucleotides and adds on
dNTP’s. In eukaryotes, a protein called replication protein A (RPA)
displaces and unwinds the RNA portion of the primer, and recruits
endonucleases to cut out the RNA portion of the primer. DNA polymerase
 Finally, ligase joins the 3’OH at the end of each DNA fragment to the 5’P
of the next fragment.
6.7 DNA sequencing
 Dideoxyribonucleotides differ from regular dNTP’s in that they are
lacking a 3’OH group. DideoxyNTP’s will effectively terminate an
elongation reaction because they can be added onto a DNA strand, but no
dNTP’s can be added to them. These chain terminators are used in DNA
sequencing, called the dideoxy sequencing method. To sequence a DNA
molecule of interest, four DNA synthesis reactions are carried out, each
one containing a dideoxy NTP for one of the four nucleotides.
o For example, the dideoxy-ATP reaction will terminate at every
adenine in the sequence. When run out on a gel, this reaction will
have fragments of lengths of bp’s where there is an A in the DNA
sequence. So, if there is a 94 basepair fragment in the dideoxyATP
reaction, there is an Adenosine at the 94th base in the strand.
 In current automated sequencing techniques, the dideoxynucleotides are
also labeled with a different fluorescent dye that allows the products to be
distinguished and detected automatically as they run off a single gel.
 Shotgun sequencing
Fragments of a genome of interest are generated and cloned into plasmid
vectors. The plasmids are sequenced. This is done multiple times randomly
so that all of the generated sequence fragments are overlapping to some
extent. These can then be put back into order according to their overlapping
sequences to generate the continuous sequence of the linear genome.
Dideoxynucleoside analogs have proven to be effective at inhibiting replication
of viral genetic material, particularly against HIV/AIDS
6.6 Recombination
Gene conversion can occur because of mismatch repair after a recombination
event. The cell mismatch repair machinery notices a non-Watson Crick base pair
in double helical DNA. A fragment of the mismatched DNA is excised from one
strand and new complementary DNA is synthesized. Therefore if the mismatch
was the result of two different alleles brought together through recombination, at
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that point in the sequence one of the alleles will win out as the sequence is made
to be complementary again.
Holliday model of recombination: no longer accepted model for recombination
but interesting for aspects of topology – resolution of the Holliday junction still
pertinent in currently accepted model
 Initiated by single stranded nicks at identical positions of same direction
strands in two DNA molecules (not likely if you think about it, unless
catalyzed by a very special protein complex that has yet to be discovered)
 Strand invasion of complementary DNA strands
 Branch migration as base pairing region migrates along strands
 Ligation of nicked portion of DNA
 Results in a four-stranded DNA molecule, must be double nicked and
rejoined to get back to two double-stranded DNA’s - junction of the 4
strands is called the Holliday junction (cf. Fig. 6.31-32)
o nick inner strands  non recombinant for flanking markers
o nick outer strands  recombinant for flanking markers
o both equally possible topologically
Double-strand break and repair model: currently favored model for
homologous recombination. (cf. Fig. 6.33)
 Initiated by a DNA molecule with an uneven double stranded break
created by exonuclease activity and a gap of missing sequence.
 Free ends of the broken duplex invade the homologous duplex and
displace one of the intact strands, forming a D loop.
 The D loop expands until it bridges the gap of missing sequence.
 Repair synthesis fills in the missing sequence and ligation creates a fourstranded molecule that will have two possible Holliday junctions.
 The junctions can be resolved in 8 different ways, half of which are
recombinant.
 There is evidence in yeast for the double-strand break and repair model of
recombination.
Chapter 7: Chromosome Organization
7.1 Genome Size and the C paradox
Bacterial genomes are usually housed in a circular DNA molecule. Bacteria also
have much smaller circular DNA molecules called plasmids that contain nonessential genes.
Eukaryotic genomes are packaged in linear structures called chromosomes.
The units of genome sizes commonly used are: kb=kilobases, 103 basepairs (bp’s)
or nucleotides (nt’s); Mb=megabase=106 bp’s or nt’s; Gb=gigabase=109 bp’s or nt’s.
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Viral genomes are usually in the range 100-1000 kb. Bacterial genomes are
usually in the range 1-10 Mb (or 1000-10,000 kb). Eukaryotic genomes typically
range from 100-1000 Mb, with some of the smallest eukaryotic genomes being 10
Mb.
The C-value paradox says that genome size does not correlate with the
metabolic, developmental and behavioral complexity of an organism.
7.2 and 7.3 DNA supercoiling and bacterial chromosomes
DNA must be compacted to fit inside the cell.
Supercoiling helps accomplish this in bacteria, it occurs when two DNA double
helices are twisted around each other. DNA that is not supercoiled is considered
to be relaxed.
Negative supercoils involves right-handed twisting of the DNA. Negative
supercoiling compensates for underwinding of DNA. Positive supercoils have
left-handed twisting of DNA and are seen in archaebacteria.
Topoisomerases govern the supercoiling of DNA. Topoisomerase I causes a
single-stranded nick in DNA, swivels it around, and reseals the broken strand to
increase or decrease the amount of supercoiling. Topoisomerase II causes a
double-stranded break in DNA to allow one double helix to pass through
another.
A single nick by DNase will relax supercoiled DNA.
Bacterial DNA is also folded up to some extent by proteins that help fold, bend
and wrap the DNA into a nucleoid structure. (cf. Fig. 7.5)
*In eukaryotes, supercoiling only occurs in a few places on a chromosome
around areas of active transcription.
7.3 The Structure of Eukaryotic Chromosomes
Eukaryotic chromosomes pack a huge length of DNA into a small space. In
Chromosome 1, an 82mm (8.2 x 104 m) length of DNA is condensed into a
chromosome 10m long and 1m wide.
Chromatin – a stable form of somewhat compacted DNA found in the cell
nucleus – DNA is complexed with 5 types of histone proteins – usually in the 30nm form that will be described below
Histones – DNA-binding proteins proteins that stabilized compacted DNA in
the cell – they are very basic, or positively charged, which allows them to bind to
negatively charged DNA and stabilize the somewhat compacted DNA
Core particle – the complex of histone proteins around which DNA is wrapped
twice. It consists of 2 molecules each of the histones H2A, H2B, H3 and H4.
Nucleosomes are the units of DNA wrapped around histone core particles,
which look like beads on a string. (cf. Fig. 7.7)
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30-nm chromatin fiber – the nucleosomes fold up into a thicker fiber that is
about 30-nm in diameter (cf. Fig. 7.9)
During interphase, the chromatin is not yet, compacted into visible
chromosomes, however there is evidence that chromatin is organized into
specific structures in specific characteristic areas of the cell.
Chromatin loops: about 100kb loops of 30nm fibers
Chromatin domains: about 1Mb each, about 300-800nm in thickness
Chromosome territory: the area of the nucleus occupied by chromatin from a
particular chromosome. It appears that chromosomes with more genes have
territories near the center of the nucleus.
Interchromatin compartment: the spaces between the chromatin fibers are
channels through which replication and transcription and RNA processing
machinery can migrate to a site of activity
Chromosome condensation – in preparation for mitosis and the movement of
genetic material into daughter cells, chromatin further condenses into
chromosomes – this occurs in stages
Scaffolding – in chromosomes, multiple loops of 30nm fibers appear to be held
together by a central non-histone protein scaffold
7.5 Polytene chromosomes
Polytene chromosomes contain around 1000 copies of DNA – they are formed
when a synapsed pair of homologous chromosomes undergoes many many
rounds of replication without any separation of the resulting molecules. The
multiple copies of the chromatin are aligned side by side, forming a very thick
chromosome which stains in identical patterns across the chromosome.
These chromosomes are found in certain tissues of insects and cells containing
them do not go through any further cell division.
These chromosomes have a staining pattern typical for each chromosome of a
given species, with dark bands in areas of greater DNA density. This staining
allows visualization of genetics markers along the chromosome, and provides a
cytological map of the chromosomes. Polytene chromosomes are useful for in
situ hybridization of a DNA or RNA probe that is fluorescently labeled and
which will be hybridized to squashed polytene DNA.