BIOL 201 Lecture Notes - Lecture 11: Replication Protein A, Restriction Enzyme, Dna Profiling
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A distinct group of DNA-binding proteins are the DNA-binding proteins that specifically bind
single-stranded DNA. In humans, replication protein A is the best-understood member of this
family and is used in processes where the double helix is separated, including DNA replication,
recombination and DNA repair These binding proteins seem to stabilize single-stranded DNA
and protect it from forming stem-loops or being degraded by nucleases.
In contrast, other proteins have evolved to bind to particular DNA sequences. The most
intensively studied of these are the various transcription factors, which are proteins that regulate
transcription. Each transcription factor binds to one particular set of DNA sequences and
activates or inhibits the transcription of genes that have these sequences close to their promoters.
The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase
responsible for transcription, either directly or through other mediator proteins; this locates the
polymerase at the promoter and allows it to begin transcription. Alternatively, transcription
factors can bind enzymes that modify the histones at the promoter; this will change the
accessibility of the DNA template to the polymerase.
As these DNA targets can occur throughout an organism's genome, changes in the activity of one
type of transcription factor can affect thousands of genes. Consequently, these proteins are often
the targets of the signal transduction processes that control responses to environmental changes
or cellular differentiation and development. The specificity of these transcription factors'
interactions with DNA come from the proteins making multiple contacts to the edges of the
DNA bases, allowing them to "read" the DNA sequence. Most of these base-interactions are
made in the major groove, where the bases are most accessible.
Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of thephosphodiester
bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are
called exonucleases, while endonucleases cut within strands. The most frequently used nucleases
in molecular biology are the restriction endonucleases, which cut DNA at specific sequences. For
instance, the EcoRV enzyme shown to the left recognizes the 6-base sequence 5′-GATATC-3′
and makes a cut at the vertical line. In nature, these enzymes
protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial
cell, acting as part of therestriction modification system. In technology, these sequence-specific
nucleases are used in molecular cloning and DNA fingerprinting.
Enzymes called DNA ligases can rejoin cut or broken DNA strands. Ligases are particularly
important in lagging strandDNA replication, as they join together the short segments of DNA
produced at the replication fork into a complete copy of the DNA template. They are also used
in DNA repair and genetic recombination.
Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the
amount of supercoiling in DNA. Some of these enzymes work by cutting the DNA helix and
allowing one section to rotate, thereby reducing its level of supercoiling; the enzyme then seals
the DNA break. Other types of these enzymes are capable of cutting one DNA helix and then
passing a second strand of DNA through this break, before rejoining the helix. Topoisomerases
are required for many processes involving DNA, such as DNA replication and transcription.
Helicases are proteins that are a type of molecular motor. They use the chemical energy
in nucleoside triphosphates, predominantly ATP, to break hydrogen bonds between bases and