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									                                                                                                                     Gettysburg College

                                                                                            Functional analysis of a novel DNA ligase in the filamentous fungus Aspergillus nidulans
                                                                                                                                                                                             Ben Woods and Steve James
                                                                                                                                                                                                                                                                                                                                    Department of Biology

Abstract                                                                                         Background
Eukaryotes possess a small number of DNA                                                         The purpose of this study is to (1) assess the functions of the four Aspergillus nidulans DNA                                                                  Deletion of AN4883-Lig3: Δ4883::pyrGAf genotypic
ligases. Mammals and most yeasts, e.g.,
possess only three DNA ligases: DNA Ligases I
                                                                                                 ligases, (2) determine the minimal set of DNA ligases for survival in a filamentous fungus, and                                                                                diagnosis by PCR
                                                                                                 (3) understand the role of the novel fourth DNA ligase, and in particular to determine if it plays a
and III (DNL1 and DNL3) are required for
                                                                                                 role in the NHEJ pathway for repair of double-strand break (DSBs).        The novel A. nidulans
chromosomal and mitochondrial DNA replication
and repair, respectively; and DNA Ligase IV
                                                                                                 DNA Ligase (AN8014) has weak homology to DNA Ligase I, AN6069 (3.35e-4), weaker                                                                   4.4
(DNL4), which is nonessential and plays a                                                        homology to DNA Ligase IV, AN0097 (e = 0.015) and is even more distantly related to DNA
specialized role in the repair of double strand                                                  Ligase III, AN4883. This divergent DNA ligase is conserved across filamentous fungi,
breaks via the Non-Homologous End Joining                                                        suggesting that it harbors a conserved function. Little is known about DNA Ligase III in                                                                                                                 2.8                              CR For
(NHEJ) pathway of DNA repair. Aspergillus                                                        filamentous fungi. However, DNL III mutations in mammals lead to embryonic lethality. DNA
                                                                                                                                                                                                                                                   2.3                                                                                                4883 – Lig3
nidulans and other filamentous fungi, however,                                                   Ligase IV has highly regulated function as the principal enzyme for the repair of DNA double-
                                                                                                                                                                                                                                                                                                                                                                      CR Rev
possess a fourth, novel DNA ligase. We have                                                      strand breaks during nonhomologous end joining (NHEJ). NHEJ is initiated by the recognition                                                       2.0                                                                                                    2.8 kb                       WT locus
deleted the novel Aspergillus DNA ligase                                                         of double strand breaks by the Ku70/Ku80 heterodimer, known as Hdf1/Hdf2 in yeast. A three-                                                                                                                                                Amplification of coding region
(AN8014) and discovered that Δ8014 strains are                                                   protein complex, Rad50/Mre11/Xrs2, is recruited to Hdf1/Hdf2 and forms a stable end-bridge for
viable and show no increases in sensitivity to                                                   the alignment of the DNA strand ends in yeast (Chen et al., 2001). Lif1 complexes with Dnl4                                                       4.4
DNA damage agents. We recently deleted DNA                                                       and assists the association of Dnl4/Lif1 to the Xrs2 subunit of Rad50/Mre11/Xrs2 (see diagram
Ligase III (DNL3), and have found that the single                                                at bottom left).                                                                                                                                                                                                                  For Nested 5’                                  Nested 3’

deletion, ΔDNL3, and double deletion, Δ8014                                                                                                                                                                                                                                                               2.7
ΔDNL3, are viable, indicating that both of these                                                       NHEJ-competent strains exhibit a high frequency of ectopic, random integration events in                                                                                                                                             2.7 kb

ligases are dispensable. Currently, we are                                                       transformation-based assays (Nayak et al., 2004). We wish to determine if the novel DNA                                                           2.3                                                                            Amplification dependent upon
                                                                                                                                                                                                                                                                                                                                                                                      After gene
testing ΔDNL3 and Δ8014 ΔDNL3 for defects in                                                     ligase (AN8014) is redundant with DNL4 for NHEJ activity. We are working to delete DNL4 and                                                       2.0                                                                               correct integration and
                                                                                                                                                                                                                                                                                                                                   replacement of Lig3 gene                           with pyrGAf
growth and DNA damage responses. In                                                              create 8014 DNL4 double mutants. Following this, we plan to use a transformation-based
addition, we are working to delete the remaining                                                 assay to measure the frequency of ectopic versus targeted integration in Δ8014, ΔDNL4, and
two DNA ligases, and to create all combinations                                                  double Δ8014 ΔDNL4 strains.
of double and triple deletions. Furthermore, we
plan to use a transformation-based assay to
measure the frequency of ectopic versus
targeted integration in Δ8014, ΔDNL4, and                                                                                  Deletion of AN8014 by replacement with the
double Δ8014 ΔDNL4 strains.                                                                                                                                         Af)
                                                                                                                           Af pyroA selectable marker (Δ8014::pyroA
          Phylogeny of Aspergillus                                                                                                                 3.9 kb
                                                                                                                                                                                                                                                                                            • The novel DNA Ligase (AN8014) is non-essential. The
                DNA ligases                                                                                          2.5 kb                                                                                                                    2.6 kb                                       deletion confers no obvious defects in growth or DNA
                                                                                                                                                                                                              2.4 kb                                                                        damage senstivity.
                                                                      AN6069                                  E P            ATG   E           P      TAG           E                            P                     E
                                                                                                                                                                                                        E P                                              E                     P
                                                                                                                                        8014                                                                               pyroAAf
                                                                                                                                                                                                                                                                                            • DNA Ligase III (AN4883) is non-essential. We are
                                                          0.0                                                                          8014 Probe                                                                      pyroAAf   probe
                                                                      AN4883                                                                                                                                                                                                                currently testing this deletion for impairment of growth and
                              3.44e-24                                                                                                                                                                                                    8.3 kb                                            response to DNA damage.
                                                                                                                            3.7 kb                              6.0 kb

                                                                                                          wild-type 8014 gene                                                                          after gene replacement                                                               • The double Δ8014 Δ4883 deletion is fully viable,
                                                                                                                                                                                                                                                                                            indicating that Aspergillus can grow normally with only two
                 3.35e-4                                                                                                                                                                                                                                                                    ligases, DNL I and DNL IV.
                                                                      AN8014                                                                        8014 coding region probe                                                  pyroAAf probe
                                                                                                                                                       Pst I                   EcoR I                                       Pst I                   EcoR I
                                                                                                                                            par par Δ1Δ2 Δ3 Δ4 Δ5       par par Δ1 Δ2 Δ3 Δ4 Δ5                   par par Δ1 Δ2 Δ3 Δ4 Δ5      par par Δ1 Δ2 Δ3 Δ4 Δ5                         • Deletion of the remaining two DNA Ligases (AN6069-
                                                                                                                                                                                                                                                                                            DNL I and AN0097- DNL IV) is underway.
                 The NHEJ pathway in budding yeast
                                                                                                                                                                                                     23 kb
                                                                                         Yeast Dnl4 pathway and possible
                                            Hdf1/Hdf2                        Hdf1/Hdf2   redundant pathway of AN8014
                                                                                                                                                                                                     9.4 kb                                                           8.3 kb

                                                           Recruitment of                                                          6.0 kb                                                            6.6 kb
                                                                                                                                                                                                     4.4 kb
                                                                                                                                   3.9 kb                                                                                                                                          This work was supported by grants to SWJ from Gettysburg College. BLW was
                                                         Rad50    Rad50
                                                                                                                                   3.7 kb                                                                                                                                          supported by the Gettysburg College Senior Project Fund. We would also like to thank
                                                  Xrs2                    Xrs2
                                                                                                                                                                                                                                                                                   Kristy Bialas, Jenna Keyser, Kristin Shingler and Martina Trovato for their technical
                                                         Mre11    Mre11                                                                                                                                                                                               2.6 kb
            Lif1 Dnl4                                                                                                                                                                                                                                                              assistance.
                                                                                                                                   2.5 kb                                                                                                                             2.4 kb
              Nej1                                 Lif1 Dnl4                                                                                                                                         2.3 kb
           Enhanced NHEJ activity
           due to Nej1                                                                                                                                                                               2.0 kb
           phosphorylation       Phosphorylated
                                                                                                                                                                                                                                                                                   Chen, L., Trujillo, K., Ramos, W., Sung, P., Tomkinson, A. 2001. Promotion of Dnl4-catalyzed DNA end-joining by
                                                  Lif1 Dnl4            Dnl4 Lif1                                                                                                                                                                                                            the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. Molecular Cell 8: 1105-1115.

                     Pol4                                                                                                                                                                                                                                                          Nayak, T., Szewczyk, E., Oakley, C.E., Osmani, A., Ukil, L., Murray, S.L., Hynes, M.J., Osmani, S.A. and Oakley,
                                                                                                                                                                                                                                                                                           B.R. 2006. A versatile and efficient gene targeting system for Aspergillus nidulans. Genetics Society of
                                                                                                                                                                                                                                                                                           America 172: 1557-1566.
                                                               Pol4   Pol4


  Lif1 Dnl4                                                                                               Xrs2
Phosphorylated                                                                                            Phosphorylated

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