MOL. BIO LEC 2 Flashcards
● Structural components that forms the outer edge of the DNA double helix composed of alternating sugar and phosphate groups
● Arranged at specific distances from one another in the double helix
● 2 regions formed in the helix by the backbones: major groove & minor groove
SUGAR - PHOSPHATE GROUP
● Often found in nature
● Base modifications have significant effects on phenotype
● Some result from DNA damage, naturally modified for specific functions or to affect gene expression
● Used by bacteria & viruses as a primitive immune system
Modified nucleotides
● Macromolecule made of nucleotides bound together by the phosphate & hydroxyl groups on their sugars
NUCLEIC ACID
DNA is oriented in ___________
5’ to 3’ direction
It is the process by which DNA makes a copy of itself during cell division
DNA REPLICATION
- every double helix in the new generation of an organism consists of 1 COMPLETE “OLD” strand and 1 COMPLETE “NEW” strand wrapped around each other
- Key to maintaining the sequence of the nucleotides in DNA through new generations
SEMI-CONSERVATIVE
Demonstrated the mechanism of semiconservative replication using the technique of equilibrium density centrifugation on a cesium gradient
MATTHEW MESELSON & FRANKLIN STAHL
POLYMERASE
- 1st purified enzyme shown to catalyze DNA replication in prokaryotes
- Followed by DNA polymerase II & |||
DNA POLYMERASE I (POL I)
POLYMERASE
- Main polymerizing enzyme during bacterial replication
- Functions as a multi-subunit HOLOENZYME
DNA POLYMERASE III (POL III)
- Addition of labeled nucleotides at nicks (single-strand breaks) in DNA
- Often used in vitro as a method to introduce labeled nucleotides into DNA molecules, which will be used for DNA detection in hybridization analyses
NICK TRANSLATION
- Type of DNA polymerase that can synthesize polynucleotide chains WITHOUT A TEMPLATE
- How?
▪ By adding nucleotide to the end of a DNA strand in the absence of H-base pairing with a template
- How?
- Used in the laboratory to generate 3’-end labeled DNA species
Terminal transferase
POLYMERASE CHARACTERISTIC:
⮚ Processivity (staying with the template longer to make longer products)
⮚ Fidelity (faithful copying of the template)
⮚ Substrate specificity (affinity for altered nucleotides)
It is the process by which cellular DNA is maintained
DNA METABOLISM
- Endonucleases that RECOGNIZE SPECIFIC BASE SEQUENCE AND BREAK OR RESTRICT THE DNA POLYMER at the sugar-phosphate backbone
- Originally isolated from bacteria, as part of a primitive immune system Named for the organism from which they were isolated:
- BamHI (from Bacillus amyloliquefaciens H)
- HindIII (from Haemophilus influenzae Rd)
- Smal (from Serratia marcescens Sbb)
RESTRICTION ENZYMES
TYPES OF RESTRICTION ENZYMES
- NUCLEASE & METHYLASE activity in a single enzyme
- Bind to host-specific DNA sites of 4-6 bp separated by 6-8 bp & containing methylated adenines
Example: Ecok from E. coli K 12, recognizes the ff site:
TYPE I RESTRICTION ENZYMES
TYPES OF RESTRICTION ENZYMES
- Used most frequently in the laboratory
- DO NOT have inherent methylation activity
- Bind as simple dimers to symmetrical 4-8 bp DNA recognition sites (palindromic)
- Cleave DNA directly at the binding site, producing fragments of predictable size
TYPE II RESTRICTION ENZYMES
TYPES OF RESTRICTION ENZYMES
- Resemble Type I enzymes in their ability to both methylate & restrict (cut) DNA
- Have multiple subunits, including helicase (unwinding) activity
- Recognition sites are asymmetrical & the cleavage of the substrate DNA occurs 24-26 bp from the site to the 3’ side
Example: Pstill from Providencia stuartii, recognizes the ff site & cuts DNA 25-26 bp 3’ to the recognition site
TYPE III RESTRICTION ENZYMES
TYPES OF RESTRICTION ENZYMES
- Similar subunit structures & enzyme requirements
- CUTTING & METHYLTRANSFERASE functions
Example: BseMII from Bacillus sterothermophilus, with target sequence:
TYPE IV RESTRICTION ENYMES
CUTS 1 strand of DNA 10 bp ff the recognition sequence.
RESTRICTION ENDONUCLEASE FUNCTION
ADDING METHYL GROUPS methyl groups to both of the adenine residues in the target sequence
METHYLATION FUNCTION
- Catalyzes the formation of a phosphodiester bond between
adjacent 3’-hydroxyl & 5’-phosphoryl nucleotide ends
(SEALS) - Discovered in 5 different laboratories in 1967
DNA LIGASE
o Degrade DNA from free 3’-hydroxyl or 5’- phosphate ends
o Used under controlled conditions, to
manipulate DNA in vitro
o Different substrate requirements & will therefore degrade specific types of DNA ends
EXONUCLEASES
degrades single-stranded DNA (ssDNA) from the 3’-hydroxyl end into mononucleotides
EXONUCLEASE I FROM E.coli
removes 5’ mononucleotides from the 3’ end of double-stranded DNA (dsDNA) in the presence of Mg2+ & Mn2+
EXONUCLEASE III FROM E. coli
