Nucleotides and Nucleic Acids - PowerPoint by 395yZ9

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									The repair of a
double-strand
break in DNA by
homologous end-
joining.


  Damaged site is
  copied from the other
  chromosome by
  recombination
  proteins
            Double strand repair
• Nonhomologous end-joining
  – only in emergency situations
  – Two broken ends of DNA are joined
    together.
  – A couple of nucleotides are cut from both
    of the strands.
  – Ligase joins the strands together.
  – Sometimes an extra nucleotide is added.
Nonhomologous End Joining
Construct design in this paper is more
 complex, but the idea is the same.
Reporter gene is GFP. Since repair is taking
  place in a eukaryotic system, ds breaks
  must be repaired, mRNAs synthesized and
  processed by normal splicing machinery to
  generate detectable levels of GFP.
SceI sites produce HR sites in Construct B
  (next slide) and NHEJ type breaks in
  construct A.
Constructs are integrated into the
  chromosomes


        Sce I site
            SD, splice donor; SA, splice acceptor;
            shaded squares, polyadenylation sites.

Supplementary Figure 1. Constructs integrated in the reporter cell lines
for detecting NHEJ and HR.
A, NHEJ reporter cassette. The construct consists of a GFP gene
containing an intron, interrupted by an adenoviral exon (Ad).The adenoviral
exon is flanked by SceI recognition sites in inverted orientation for
induction of DSBs. Inverted orientation prevents overlap of sticky ends
produced by SceI cleavage (see Figure 1C).

 In the starting construct the GFP gene is inactive. Induction of a DSB by
I-SceI followed by NHEJ, transcription and splicing reconstitutes the
functional GFP gene.
SceI recognition site




…TAGGGATAACAGGGTAAT…ATC CCTATTGTCCC ATT…
…ATCCC TATT GTCCCATTA…TAGGGATAACAGGGTAAT




        Two SceI sites in inverted orientation
        make incompatible sticky ends.
B, HR reporter cassette. The construct consists of two mutated copies of
GFP-Pem1. In the first copy of GFP-Pem1 the first GFP exon carries a
deletion of 22 nt and an insertion of two I-SceI recognition sites in
inverted orientation. The 22 nt deletion ensures that GFP cannot be
reconstituted by an NHEJ event. The second copy of GFP-Pem1 is lacking
the ATG (for translation initiation) and the second exon of GFP. Upon
induction of DSBs by I-SceI, gene conversion events reconstitute the GFP
gene.
     The construct consists of two mutated copies of
     GFP-Pem1. In the first copy of GFP-Pem1 the
     first GFP exon carries a deletion of 22 nt and an
     insertion of two I-SceI recognition sites in
     inverted orientation. The 22 nt deletion ensures
     that GFP cannot be reconstituted by an NHEJ
     event. The second copy of GFP-Pem1 is lacking
     the ATG and the second exon of GFP. Upon
     induction of DSBs by I-SceI, gene conversion
     events reconstitute the GFP gene.



#1
                   #2
                                           Fig. 1 SIRT6 stimulates DSB repair.




Published by AAAS   Z Mao et al. Science 2011;332:1443-1446
                               Fig. 1 SIRT6 stimulates DSB repair.




       A) Overexpression of SIRT1, -2, -6, and -7 in human fibroblasts.
       Immunoblotting with sirtuin-specific antibodies after transfection
       with a sirtuin-expressing vector or a control vector encoding
       hypoxanthine-guanine phosphoribosyltransferase (pControl).

      Z Mao et al. Science 2011;332:1443-1446


                       Z Mao et al. Science 2011;332:1443-1446



Published by AAAS
   Fig. 1 SIRT6 stimulates
          DSB repair.




(B) Effect of sirtuin overexpression on the efficiency of
NHEJ and HR, measured as described in (27) and fig. S1. The
efficiency of DSB repair was scored in untreated cells (open
bars), cells pretreated with 1 mM paraquat for 16 hours
(black bars), or cells treated with paraquat and 5 mM
nicotinamide for 16 hours (red bars).
Error bars indicate SD; n = 8 experiments (control and SIRT6); n = 3 (other sirtuins). P values were calculated by two-tailed
                                 Z Mao et al. Science 2011;332:1443-1446
Student’s t test..


Published by AAAS
  Fig. 1 SIRT6 stimulates DSB repair.




(C) SIRT6 overexpression accelerates the
disappearance of γH2AX foci after treatment with 1
mM paraquat for 16 hours. Data represents an average
of at least 50 nuclei.
                               Fig. 1 SIRT6 stimulates DSB repair.


              (D) Immunoblot showing induction
              of endogenous SIRT6 protein levels
              by oxidative stress. Human
              fibroblasts were treated with
              paraquat for 16 hours.




      Z Mao et al. Science 2011;332:1443-1446


                        Z Mao et al. Science 2011;332:1443-1446



Published by AAAS
                    Fig. 2 Oxidative stress results in early recruitment of SIRT6 to DNA breaks.




  Oxidative stress results in early recruitment of SIRT6 to DNA breaks. ChIP analysis
  showing kinetics of SIRT6 recruitment to Alu sequences after 8 Gy of γ-irradiation (IR)
  (A) and sequences flanking I-SceI–induced DSB after transfection with I-SceI expression
  vector

  (B). Asterisks indicate values significantly different from corresponding zero time points (P
  < 0.05). Error bars indicate SD; n = 5. Control ChIP with SIRT6−/− cells is shown in fig. S7.
  IgG, immunoglobulin G.
         Z Mao et al. Science 2011;332:1443-1446



Published by AAAS
      Fig. 3 Deacetylation and mono-ADP-ribosylation activities of SIRT6 are required to stimulate
                                            DSB repair.




(A)   Immunoblot showing that S56Y and R65A
      mutations abolish the H3K9 deacetylation activity
      of Sirt6 and appear to exert a dominant-negative
      effect.

(B) In vitro assay showing that S56Y and G60A
   mutations abolish mono-ADP-ribosylation activity
   of SIRT6. NAD+, nicotinamide adenine
   dinucleotide.




                                                                      Z Mao et al. Science 2011;332:1443-1446
Fig. 3 Deacetylation and mono-ADP-ribosylation activities of SIRT6 are required to stimulate
                                      DSB repair.




(A) Immunoblot showing that S56Y and R65A
    mutations abolish the H3K9 deacetylation
    activity of Sirt6 and appear to exert a
    dominant-negative effect.
(B) In vitro assay showing that S56Y and G60A
    mutations abolish mono-ADP-ribosylation
    activity of SIRT6. NAD+, nicotinamide
    adenine dinucleotide.
(C) SIRT6 mutants for deacetylation and/or
    ribosylation activities have reduced ability to
    stimulate NHEJ and HR. Untreated cells
    (open bars) or cells treated with paraquat
    (black bars) were transfected with SIRT6-
    expressing vectors or pControl.

                                                                Z Mao et al. Science 2011;332:1443-1446
        Fig. 4 SIRT6 interacts with PARP1 and stimulates its poly-ADP-ribosylation activity.

                                                                     Z Mao et al. Science 2011;332:1443-1446




(A) Analysis of mono-ADP-ribosylated
    proteins in the WT and SIRT6−/− MEFs
    stressed with paraquat for 16 hours.
    Cells were transfected with biotinylated
    NAD, and poly-ADP-ribosylated proteins
    were cleared away with PAR antibodies.

(B) Immunoblotting of the extracts in (A)
    with PARP1 antibodies indicated that the
    120-kD band is Parp1.

(C) SIRT6 interacts with PARP1. Human
    fibroblasts were treated with 1 mM
    paraquat. Cell lysates were
    immunoprecipitated with SIRT6
    antibodies in the presence of ethidium
    bromide (EtBr) followed by Western
    blotting with PARP1 antibodies.
(D) PARP1 K521 is essential for activation   (E) PARP1 lacking the catalytic domain is
of NHEJ by SIRT6. An NHEJ assay was          mono-ADP-ribosylated by SIRT6 in vitro,
performed in PARP1−/− MEFs containing an     whereas K521A is not.
integrated NHEJ reporter. Cells were
transfected with SIRT6 and/or PARP1 or
PARP1 mutants. Both SIRT6 and PARP1 are      (F) In vitro assay of PARP1 poly-ADP-
required for the stimulation of repair.      ribosylation activity showing that PARP1 is
PARP1 Y889C is a catalytically inactive      stimulated only by SIRT6 mono-ADP-
PARP1. PARP1 DEEKKK contains mutations       ribosylation activity. (G) Stimulation of
in all six poly-ADP-ribosylation sites.      NHEJ and HR by SIRT6 is abolished by
PARP1 DEEKK contains the same                PARP1 inhibitors 3-ABA or PJ34.
mutations, except at K521. Asterisks
indicate values significantly different
from control (P < 0.01).
(D) PARP1 K521 is essential for activation   (F) In vitro assay of PARP1 poly-ADP-
of NHEJ by SIRT6. An NHEJ assay was          ribosylation activity showing that
performed in PARP1−/− MEFs containing an     PARP1 is stimulated only by SIRT6
integrated NHEJ reporter. Cells were         mono-ADP-ribosylation activity.
transfected with SIRT6 and/or PARP1 or
PARP1 mutants. Both SIRT6 and PARP1 are      (G) Stimulation of NHEJ and HR by
required for the stimulation of repair.      SIRT6 is abolished by PARP1 inhibitors
PARP1 Y889C is a catalytically inactive      3-ABA or PJ34.
PARP1. PARP1 DEEKKK contains mutations
in all six poly-ADP-ribosylation sites.
PARP1 DEEKK contains the same
mutations, except at K521. Asterisks
indicate values significantly different
from control (P < 0.01). (E) PARP1 lacking
the catalytic domain is mono-ADP-
ribosylated by SIRT6 in vitro, whereas
K521A is not.
Binding of Proteins to DNA Often
   Involves Hydrogen Bonding

      Types of domains that bind DNA:

      Helix-turn-helix
      Zinc Finger
      Leucine Zipper
      Helix-loop-helix
Functional
groups on all
four base pairs
that are
displayed in the
major and minor
grooves of DNA
A = H bond acceptor

D = H bond donor
Guanine-Arginine: One of the most common
DNA-protein interactions.

Because of its specific
geometry of H-bond acceptors, guanine
can be unambiguously recognized by the
side chain of arginine
 Helix-Turn-Helix Motif is Common
      in DNA-Binding Proteins

• One of the helixes (red) fits into the major groove of
  DNA
• Four DNA-binding helix-turn-helix motifs (gray) in
  the Lac repressor
Important Points: a handshake leads to a bear-hug


 Specific recognition of DNA targets by the helix-turn-helix motif
 involves interactions between sides of the recognition helix and
 bases in the major groove of the DNA

But, specific recognition of DNA sequences is to a large
extent governed by other interactions within
complementary surfaces between the protein and the
DNA


These interactions frequently involve H-bonds from protein main-
chain atoms to the DNA backbone in both the major and the minor
groove and are dependent on the sequence-specific deformability of
the target DNA
Helix-turn-helix. (a) DNA-binding domain of the Lac repressor




                              The helix-turn-helix motif is
                              shown in red and orange; the
Helix-turn-
helix. (c)
Surface
rendering of the
DNA-binding
domain of the
Lac repressor
bound to DNA.
The DNA-binding domain separated from the DNA, with the
binding interaction surfaces shown.
groups on the protein and DNA that interact through H-bonding
groups that interact through hydrophobic interactions
           Chapter 8: Summary
In this chapter, we learned about:

•   Function of nucleotides and nucleic acids
•   Names and structures of common nucleotides
•   Structural basis of DNA function
•   Reversible denaturation of nucleic acids
•   Chemical basis of mutagenesis
                                                                  Hexons and
        (TP) Covalently linked to DNA
                                                                  pentons form
                                                                  capsid




             36 kbp

50 nm




                         Transcribed by RNA pol II




                                                          Transcribed by RNA pol III


                                            Figure A-1 Adenovirus

								
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